MULTI-AMINOACYL-TRNA SYNTHETASE COMPLEXES IN

ARCHAEAL TRANSLATION

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Corinne D. Hausmann, B.S.

*****

The Ohio State University 2008

Dissertation Committee:

Professor Michael Ibba, Advisor Approved By

Professor Juan Alfonzo

Professor Charles Daniels ______

Professor Kurt Fredrick Advisor

Graduate Program in Microbiology

ABSTRACT

Translation, the process of mRNA-encoded protein synthesis, requires aminoacyl-tRNA synthetases (aaRSs), which function to link amino acid and cognate tRNA molecules. Once synthesized, elongation factor 1 alpha (EF-1α, the archaeal and eukaryotic homologue of the bacterial EF-Tu) binds and ushers aminoacyl-tRNAs to the ribosome during protein synthesis. To perform the essential task of aminoacylation, aaRSs must correctly bind a diverse array of molecules, including ATP, amino acids, tRNAs, and in some cases additional protein factors. For example, several eukaryotic aaRSs are known to associate with EF-1α or additional aaRSs forming higher order complexes, although the role that many of these multi-enzyme complexes play in translation remains largely undefined.

To explore the possible broader role of such aaRS complexes in translation, a

Methanothermobacter thermautotrophicus cDNA library was screened for proteins interacting with three candidate translational proteins as bait: lysyl-tRNA synthetase

(LysRS), prolyl-tRNA synthetase (ProRS), and EF-1α. Each of the three yeast two- hybrid screens revealed an interaction with M. thermautotrophicus leucyl-tRNA synthetase (LeuRS), suggesting that all four proteins may associate in vivo. Indeed, biophysical analyses confirmed the formation of a higher order multi-aminoacyl-tRNA

ii synthetase complex (MSC) in archaea, composed of LysRS, LeuRS, ProRS, and

EF-1α.

The functional consequences of complex formation were investigated by determining the steady-state aminoacylation parameters of each aaRS alone or in the

MSC. In the presence of LeuRS, the catalytic efficiencies of aminoacylation by LysRS and ProRS were enhanced three-fold and five-fold, respectively, while no significant changes in the kinetics of aminoacylation by LeuRS were observed. Furthermore, the

Leu presence of EF-1α resulted in an eight-fold increase in the kcat of Leu-tRNA synthesis, while only modest effects on the activities of EF-1α were observed. Taken together, the cellular association of archaeal LysRS, LeuRS, ProRS, and EF-1α into a higher order MSC may promote the efficiency of translation both by enhancing the aminoacylation activities of the three associated aaRSs and by coupling two stages of translation: aminoacylation of cognate tRNAs and their subsequent channeling to the ribosome.

iii

Dedicated to my parents, Guy and Cynthia, who have supported me all these years…

iv

ACKNOWLEDGMENTS

I wish to thank my advisor, Dr. Michael Ibba, for his support, encouragement, generosity, and never-ending patience throughout my graduate career and during the completion of this dissertation. It has been an honor and a pleasure working with and learning from such a talented mentor, who has contributed tremendously to my development as a scientist.

I would like to thank my committee members, Dr. Juan Alfonzo, Dr. Charles

Daniels, and Dr. Kurt Fredrick, for their insight, time, and scientific discussions throughout the years.

I am forever thankful to have met and worked with Dr. Sandro Ataide, Jiqiang

Ling, and Noah Reynolds. Their support, friendship, and interesting discussions

(scientific or otherwise) have made my graduate career enjoyable.

I wish to thank the past and present members of the Ibba lab for providing insight, support, and critical review. I would like to thank Mette Prætorius-Ibba for scientific insight and patience when I initially began this project

This work was supported by grants from the National Institutes of Health.

v

VITA

February 18, 1981 ...... Born - Seoul, Korea

2002...... B.S., Biology, Virginia Polytechnic Institute and State University

2003...... Graduate Teaching and Research Associate, The Ohio State University

PUBLICATIONS

1. Hausmann, C.D. & Ibba, M. (2008) Structural and functional mapping of the archaeal multi-aminoacyl-tRNA synthetase complex. FEBS Lett 582, 2178-2182.

2. Hausmann, C.D. & Ibba, M. (2008) Aminoacyl-tRNA synthetase complexes: molecular multitasking revealed. FEMS Microbiol Rev, in press.

3. Hausmann, C.D., Prætorius-Ibba, M., & Ibba, M. (2007) An aminoacyl-tRNA synthetase:elongation factor complex for substrate channeling in archaeal translation. Nucl Acids Res 35, 6094-6102.

4. Hausmann, C.D., Ling, L. & Ibba, M. (2007) The unnatural culture of amino acids. Nat Methods 4, 205-206.

5. Prætorius-Ibba, M., Hausmann, C.D., Paras, M., Rogers, T.E. & Ibba, M. (2007) Functional association between three archaeal aminoacyl-tRNA synthetases. J. Biol Chem 282, 3680-3687.

6. Prætorius-Ibba, Ataide, S.F., Hausmann, C.D., Levengood, J.D., Ling, J., Wang, S., Roy, H. & Ibba, M. (2006) Quality Control during aminoacyl-tRNA synthesis. Kem Ind 55, 129-134.

vi FIELDS OF STUDY

Major Field: Microbiology

vii

TABLE OF CONTENTS

Page

Abstract...... ii Dedication...... iv Acknowledgments...... v Vita...... vi List of Tables ...... xi List of Figures...... xiii List of Symbols/Abbreviations ...... xv

Chapters:

1. Introduction...... 1

1.1. The ribosome and protein synthesis...... 2 1.2. Two classes of aminoacyl-tRNA synthetases...... 5 1.3. Quality control of protein synthesis...... 7 1.4. Bacterial aminoacyl-tRNA synthetase complexes in translation...... 15 1.5. Aminoacyl-tRNA synthetase complexes in Saccharomyces cerevisiae...... 21 1.6. Mammalian multi-aminoacyl-tRNA synthetase complexes in translation .....27 1.7. Aminoacyl-tRNA synthetase complexes in Homo sapiens ...... 34 1.8. Association of aaRSs with translation EF-1α...... 40 1.9. Multi-aminoacyl-tRNA synthetase complexes in archaea...... 44

2. Chapter 2...... 46

2.1. Introduction...... 46 2.2. Materials and Methods...... 47 2.2.1. LysRS protein purification...... 47 2.2.2. ProRS and LeuRS protein purification ...... 49 2.2.3. Methanothermobacter thermautotrophicus total tRNA purification...51 2.2.4. Co-purification of native LysRS, LeuRS, and ProRS via size exclusion and anion exchange column chromatography...... 52 2.2.5. Co-purification of His-tagged LysRS, LeuRS, and ProRS by size exclusion chromatography...... 53 2.2.6. Fluorescent labeling of LeuRS...... 53 2.2.7. Fluorescence anisotropy experiments: LeuRS with ProRS and LysRS...... 54

viii 2.2.8. Preparation of in vitro transcribed tRNA...... 55 2.2.9. Aminoacylation assays for archaeal LysRS, LeuRS, and ProRS ...... 56 2.3. Results...... 57 2.3.1. Yeast two-hybrid analysis of proteins interacting with LysRS and ProRS ...... 57 2.3.2. Association of LysRS, LeuRS, and ProRS in vivo and in vitro...... 60 2.3.3. Determination of dissociation constants within the archaeal multi-aminoacyl-tRNA synthetase complex...... 64 2.3.4. Effects of association of LysRS, LeuRS, and ProRS on aminoacylation...... 65 2.4. Discussion...... 67 2.4.1. Identification of LysRS- and ProRS-interacting proteins via yeast two-hybrid analysis...... 67 2.4.2. Functional consequences of aaRS complexes in translation...... 68

3. Chapter 3...... 72

3.1. Introduction...... 72 3.2. Materials and Methods...... 73 3.2.1. Yeast two-hybrid strains, plasmid construction, and cDNA library....73 3.2.2. Yeast two-hybrid screening ...... 74 3.2.3. Archaeal EF-1α, AlaRS, and bacterial EF-Tu protein production and purification ...... 76 3.2.4. In vitro transcription of tRNA...... 77 3.2.5. Aminoacylation assays...... 78 3.2.6. Preparation of Leu-tRNALeu ...... 78 3.2.7. Fluorescence anisotropy experiments: EF-1α with LeuRS and AlaRS...... 79 3.2.8. Co-immunoprecipitation of EF-1α with LeuRS and AlaRS ...... 80 3.2.9. Nucleotide exchange and GTP hydrolysis by EF-1α ...... 81 3.2.10. Elongation factor protection of aminoacyl-tRNA against spontaneous deacylation ...... 82 3.2.11. Co-purification of LeuRS, LysRS, ProRS, and detection of EF-1α by immunoblotting...... 82 3.3. Results...... 83 3.3.1. Identification of proteins interacting with EF-1α...... 83 3.3.2. Association of EF-1α with LeuRS and AlaRS ...... 86 3.3.3. Effects of the EF-1α·LeuRS complex on GTP hydrolysis by EF-1α..90 3.3.4. Effects of complex formation between EF-1α and LeuRS on aminoacylation...... 93 3.3.5. Association of EF-1α with the multi-aminoacyl-tRNA synthetase complex...... 95

ix 3.4. Discussion...... 99 3.4.1. Identification of proteins interacting with archaeal EF-1α...... 99 3.4.2. Effects of complex formation on the aminoacylation activities of the archaeal multi-aminoacyl-tRNA synthetase complex...... 100

4. Chapter 4...... 103

4.1. Introduction...... 103 4.2. Materials and Methods...... 106 4.2.1. Protein production and purification of LeuRS mutants ...... 106 4.2.2. Fluorescent labeling of translational proteins...... 109 4.2.3. Fluorescence anisotropy experiments...... 110 4.2.4. Surface plasmon resonance experiments ...... 111 4.2.5. Preparation of in vitro transcribed tRNA...... 112 4.2.6. Activation of EF-1α and bacterial EF-Tu...... 113 4.2.7. ATP consumption editing assay...... 113 4.3. Results...... 114 4.3.1. Defining the roles of LeuRS N- and C-termini on complex formation...... 114 4.3.2. Effects of the LeuRS CP1 editing domain on complex formation ....118 4.3.3. Effects of LeuRS:EF-1α complex formation on editing by LeuRS ..123 4.4. Discussion...... 123 4.4.1. Examination of the role LeuRS plays in the formation of the archaeal multi-aminoacyl-tRNA synthetase complex ...... 123 4.4.2. Functional association of LysRS, LeuRS, ProRS, and EF-1α...... 125

5. Conclusions...... 134

Reference List ...... 140

x

LIST OF TABLES

Table Page

1.1 Classes of aaRSs and their oligomeric states...... 6

1.2 AaRSs known to possess editing activities and their non-cognate substrates ...... 9

1.3 The cis- and trans-editing factors of aaRSs...... 12

1.4 AaRS complexes in bacteria ...... 16

1.5 AaRS complexes in eukaryotes ...... 23

1.6 Non-canonical activities of aaRSs beyond translation...... 35

2.1 Yeast two-hybrid interactions: LeuRS with ProRS and LysRS...... 58

2.2 Proteins identified as interacting with ProRS ...... 59

2.3 Proteins identified as interacting with LysRS...... 59

2.4 Steady-state aminoacylation kinetics of M. thermautotrophicus ProRS ...... 66

2.5 Steady-state aminoacylation kinetics of M. thermautorophicus LysRS...... 66

2.6 Steady-state aminoacylation kinetics of M. thermautotrophicus LeuRS...... 67

3.1 Yeast two-hybrid interactions: EF-1α with LeuRS and AlaRS...... 85

3.2 Proteins identified as interacting with M. thermautotrophicus EF-1α ...... 86

3.3 Steady-state aminoacylation kinetics of M. thermautotrophicus LeuRS...... 93

4.1 Determination of LeuRS concentrations...... 116

4.2 Fluorescence anisotropy experiments: Full-length LeuRS and LeuRS

N-terminal truncations ...... 117

xi 4.3 Fluorescence anisotropy experiments: LeuRS C-terminal truncations...... 118

4.4 Fluorescence anisotropy experiments: Deletion of the LeuRS CP1

editing domain ...... 119

xii

LIST OF FIGURES

Figure Page

1.1 Overview of translation...... 3

1.2 The aminoacylation reaction performed by aaRSs ...... 5

1.3 Schematic of aminoacylation and co-translational insertion of tRNA in response

to a particular codon...... 7

1.4 Schematic for the editing of non-cognate amino acid substrates by aaRSs...... 10

1.5 Schematic of the double-sieve editing mechanism by IleRS...... 11

1.6 Crystal structure of Pyrococcus horikoshii LeuRS...... 14

1.7 Two-step pathways for the formation of Asn-tRNAAsn and Gln-tRNAGln ...... 18

1.8 Schematic diagram of the mammalian multi-aaRS complex...... 29

1.9 Schematic of translational silencing by the GAIT complex ...... 37

1.10 Schematic of the events in HIV-1 reverse transcription ...... 39

1.11 The two consecutive roles aaRSs and EF-1α play in translation ...... 43

2.1 Co-purification of LysRS, LeuRS, and ProRS ...... 62

2.2 Gel filtration chromatography of His-tagged LysRS, LeuRS, and ProRS ...... 63

2.3 Fluorescence anisotropy experiments: LeuRS with ProRS and LysRS...... 65

2.4 Multi-aaRS complexes in H. sapiens and M. thermautotrophicus ...... 69

3.1 Co-immunoprecipitation of EF-1α with LeuRS and AlaRS ...... 88

3.2 Fluorescence anisotropy experiments: EF-1α with LeuRS and AlaRS...... 89

xiii 3.3 Protection of Leu-tRNALeu by archaeal EF-1α and bacterial EF-Tu ...... 91

3.4 Effects of complex formation on GTP hydrolysis by EF-1α...... 92

3.5 Aminoacylation by LeuRS in the presence of archaeal EF-1α and

bacterial EF-Tu ...... 94

3.6 Co-purification of LysRS, LeuRS, ProRS, and EF-1α...... 96

3.7 Effects of the association of EF-1α and the archaeal multi-aminoacyl-tRNA

synthetase complex on the aminoacylation activities of LeuRS, LysRS,

and ProRS ...... 98

4.1 Schematic of the M. thermautotrophicus LeuRS amino acid sequence ...... 115

4.2 Surface plasmon resonance experiments ...... 121

4.3 ATP consumption assays ...... 122

4.4 Crystal structure of Pyrococcus horikoshii LeuRS in the aminoacylation

state ...... 127

4.5 Structural modeling of a P. horikoshii LeuRS:T. aquifex EF-Tu complex ...... 130

4.6 Crystal structure of P. horikoshii...... 132

4.7 Diagram of the proposed structural and functional association between

LysRS, LeuRS, ProRS, and EF-1α...... 133

xiv

LIST OF SYMBOLS/ABBREVIATIONS

3-AT 3-aminotriazole

5-FOA 5-fluoroorotic acid

Å angstrom (unit) aa amino acid aaRS aminoacyl-tRNA synthetase

(three letter amino acid code followed by the suffix RS) aa-tRNAaa aminoacyl-tRNA

Ala alanine

AMP adenosine 5’-monophosphate

Arg arginine

Asp aspartic acid

ATP adenosine 5’-triphosphate

ºC degree Celsius (centrigrade)

Cys cysteine

∆ delta (deletion)

Da daltons

DNA deoxyribonucleic acid

DTT dithiothreitol

xv EDTA ethylenediaminetetracetic acid

EF-1α elongation factor 1 alpha

EF-Tu elongation factor Tu g gram

Gln glutamine

Glu glutamic acid xg times gravitational constant h hour (unit)

HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid])

Ile isoleucine

IPTG isopropyl-β-D-thiogalactoside kDa kilodalton (unit)

LB Luria-Bertani

Leu leucine

Lys lysine m mili

M molar

µ micro min minute

Met methionine mRNA messenger ribonucleic acid

MW molecular weight

PAGE polyacrylamide gel electrophoresis

xvi PCR polymerase chain reaction

PEG polyethylene glycol

Phe phenylalanine

PPi inorganic pyrophosphate

Pro proline

RNA ribonucleic acid s second

SDS sodium dodecyl sulfate

Tris-HCl tris-(hydroxylmethyl) aminomethane hydrochloride tRNA transfer ribonucleic acid

Trp tryptophan

Tyr tyrosine

Val valine v volume w weight

xvii

CHAPTER 1

INTRODUCTION

The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is critical for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNA

(aa-tRNA). To carry out the essential task of aminoacylation, aaRSs must correctly bind a diverse array of molecules, including ATP, amino acids, and tRNA. In some cases, aaRSs are also known to associate with additional or auxiliary protein factors.

These associated proteins may be primarily employed in the cellular task of translation or in other processes, expanding the repertoire of functions aaRSs may perform.

Although first described in mammalian cells with the discovery of a macromolecular aaRS complex containing nine aminoacylation activities (see section

1.6), a number of multi-enzyme complexes harboring aaRSs have been described in all three domains of life. The biological role these complexes play in translation, however, remains largely undefined. This work investigates the functional association of a complex discovered in the archaeal species Methanothermobacter thermautotrophicus composed of elongation factor (EF)-1α, lysyl-, leucyl-, and prolyl-tRNA synthetases

(LysRS, LeuRS, and ProRS, respectively). As all three aaRS activities are found in the

1 mammalian macromolecular complex, the functional association of aaRSs in archaea provides further insight into the role these complexes play in translation.

1.1. The ribosome and protein synthesis

The translation of mRNA into protein is faithfully perpetuated by the ribosome, a macromolecular machine formed by the joining of two ribosomal subunits (small and large) composed of both RNA and protein components (Cate et al., 1999; Korostelev &

Noller, 2007; Steitz, 2008; Takyar et al., 2005). To accomplish the fundamental task of protein synthesis, a carefully orchestrated series of events occur on the ribosome, which may be divided into three main stages: initiation, elongation, and termination/ribosome recycling. Each stage requires the intricate coordination of the ribosome, RNA, and several protein components including translation initiation factors (IFs), elongation factors (EFs), and aaRSs. Although the nomenclature and number of protein factors involved in translation differ in all three domains of life, the essential steps of translation remain the same (Kapp & Lorsch, 2004; Kavran & Steitz, 2007). A brief overview of these three stages is presented below (Figure 1.1).

2

Large ribosomal sub. Ribosome recycling mRNA

Small sub. Large ribosomal sub. RRFs Initiation Meti + Small sub. Initiation factors Initiation RRFs factors Release Meti factors Release factors Termination Start

EF-1α (EF-T u) GTP EF-1α (EF-T u) GDP Elongation Elongation factors Meti

Elongation factors Stop

Figure 1.1. Overview of translation. In all three domains of life, translation may be divided into three main stages: initiation, elongation, and termination/ribosome recycling. Initiation involves the proper positioning of the ribosome and initiator tRNA at the mRNA start codon, accomplished with the help of several initiation factors. Once assembled at the start codon, translation elongation proceeds, in which EF-1α (the archaeal and eukaryotic homologue of the bacterial EF-Tu) binds and delivers aminoacyl-tRNA to the ribosome based on the corresponding mRNA codon. Peptide transfer and translocation of the mRNA with respect to the ribosome follows, and this process continues until a stop codon is reached, and the full-length protein dissociates. Finally, the ribosomal subunits dissociate with the help of ribosome recycling factors (RRFs) and prepare for the next round of translation.

3 Translation initiation involves joining of the small and large ribosomal subunits, proper assembly at the mRNA start codon, and correct positioning of the initiator aa- tRNA within the ribosomal peptidyl site (P site), which is accomplished with the aid of several IFs (Antoun et al., 2006a; Antoun et al., 2006b; Aravind & Koonin, 2000;

Korostelev & Noller, 2007; Kozak, 1999; Laursen et al., 2005; Lovmar & Ehrenberg,

2006). Selection and positioning of initiator tRNA in the ribosomal P site requires specific base-pairing of the initiator tRNA anti-codon with the mRNA triplet start codon. Once correctly assembled at the mRNA start site, the ribosome is competent for protein synthesis and translation elongation may proceed.

The polypeptide chain is elongated following translation initiation by the subsequent decoding of mRNA triplet codons in the 5’ to 3’ direction (Andersen et al.,

2003; Nilsson & Nissen, 2005). EF-1α (the archaeal and eukaryotic equivalent of the bacterial EF-Tu) functions to deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosomal acceptor site (A site) as the ternary complex aa-tRNA·EF-1α·GTP (Asahara &

Uhlenbeck, 2005; Dale et al., 2004; Gromadski et al., 2007; Inagaki et al., 2003;

LaRiviere et al., 2001; Pingoud et al., 1977). Upon codon-anticodon recognition GTP is hydrolyzed, which allows for accommodation of the aa-tRNA into the A site. The inactive EF-1α·GDP dissociates from the ribosome and is activated for the next round of aa-tRNA selection by the nucleotide exchange factor EF-1β (homologous to the bacterial EF-Ts) that facilitates the exchange of GDP for GTP.

Following peptide transfer of the amino acid chain from the P site to the A site

(elongating the polypeptide chain by one amino acid), translocation of the peptidyl- and acceptor-tRNAs within the ribosome is promoted by elongation factor 2 (EF-2, the 4 archaeal and eukaryotic homologue of the bacterial EF-G) (Fraser & Hershey, 2005;

Hansen et al., 2002; Horan & Noller, 2007; Moore & Steitz, 2003; Nissen et al., 2000;

Peske et al., 2004). This provides an empty A site, which may then accept an aa-tRNA molecule from EF-1α based on the next codon of the mRNA message. Delivery of aa- tRNA to the A site and subsequent translocation proceeds until an mRNA stop codon is reached, whereby the mRNA and full-length protein are released by release factors and the ribosomal subunits dissociate (Barat et al., 2007; Peske et al., 2005).

1.2. Two classes of aminoacyl-tRNA synthetases

The faithful translation of mRNA into protein requires aaRSs, which provide the elongating polypeptide chain with amino acid substrates in the form of aa-tRNAs.

The aaRSs are responsible for accurately attaching the correct amino acid onto the cognate tRNA molecule in a two-step reaction (Figure 1.2) (Ibba & Soll, 2004).

aa + aaRS + ATP aaRS • aa-AMP + PPi (1)

aaRS • aa-AMP + tRNA aaRS + aa-tRNA + AMP (2)

Figure 1.2. The aminoacylation reaction performed by aaRSs. Aminoacylation proceeds as a two-step reaction in which the amino acid is activated with ATP and subsequently transferred to the tRNA 3’-end.

Once bound in the active site, the α-carboxylate of the amino acid attacks the α- phosphate of ATP, forming an enzyme-bound aminoacyl-adenylate (aa-AMP)

5 intermediate and an inorganic phosphate leaving group. Once activated, the 2′- or

3′-hydroxyl of the terminal ribose of the corresponding tRNA nucleophilically attacks the aa-AMP, leading to the formation of aa-tRNA and an AMP leaving group.

Although related by this common mechanism of aminoacylation, aaRSs may be separated into two classes (I and II) based on the structural topologies of their active sites (Table 1.1; (Cusack et al., 1990; Eriani et al., 1990).

Class I Class II

ValRS (α)SerRS(α2) LeuRS (α)ThrRS(α2) IleRS (α)GlyRS(α2) ArgRS (α)ProRS(α2) CysRS (α)HisRS(α2) MetRS (α)AspRS(α2) GluRS (α)AsnRS(α2) LysRS I (α)LysRSII (α2) GlnRS (α)GlyRS(αβ)2 TrpRS (α)PheRS[(αβ)2, α] TyrRS (α)AlaRS(α, α4) PylRS1 SepRS1

Table 1.1. Classes of aaRSs and their oligomeric states. 1Oligomeric state is undefined.

Class I aaRSs are generally monomeric and composed of a Rossmann- nucleotide binding fold, containing the highly conserved sequence elements KMSKS and HIGH. Class I enzymes approach tRNA from the minor groove of the tRNA acceptor stem, aminoacylating the terminal adenosine of the tRNA molecule at the

2’-OH position. In contrast, the active sites of class II aaRSs are composed of

6 anti-parallel β-sheets and are generally multimeric enzymes. Class II aaRSs approach

the major groove of their respective tRNAs and couple the amino acid to the 3’-OH of the terminal adenosine. Once synthesized, aa-tRNAs are bound by EF-1α as a ternary

complex with GTP and delivered to the ribosome during translation elongation (Figure

1.3).

Val Arg Met Asn Thr Gln Tyr Ala Lys PheGlu Asp Gly Trp Pro Ile Leu Leu ATP AMP + PPi Leu GTP GDP + Pi Ser Cys His

Leucyl-tRNA synthetase Elongation factor AAG AAG 3’ UUC 5’ Leu-tRNALeu mRNA AAG Ribosome

Figure 1.3. Schematic of aminoacylation and co-translational insertion of tRNA in response to a particular codon. Adapted from Ibba & Soll, 2001.

1.3. Quality control of protein synthesis

Faithful translation of mRNA into protein relies on the accuracy of two

processes: accurate recognition of the mRNA codon by the corresponding tRNA

anticodon on the ribosome and the coupling of correct amino acid:tRNA pairs. The central role of aaRSs in defining the genetic code places a strong selective pressure on these enzymes to prevent mistakes during cognate aa-tRNA formation. The specificity with which aaRSs recognize cognate tRNAs is dependent on the contribution of both thermodynamic and kinetic effects. The aaRSs recognize their cognate tRNA

7 molecules based on certain characteristics of the tRNA structure or due to the presence

of modified or unmodified nucleotides at particular positions, creating a unique set of

identity elements for each aaRS:tRNA pair. These identity elements are often located

in the tRNA acceptor and anticodon stems, the anticodon loop, and the tRNA variable

arm. It has also been suggested that specific mutations within the tRNA molecule, such

as in the acceptor stem, greatly effect the kinetics of aminoacylation (kcat) while only modestly influencing binding (KM) to the cognate aaRS (Guth & Francklyn, 2007;

Kelley et al., 2000). Although the mechanism is not completely understood, this

indicates a kinetic proofreading mechanism by the aaRSs, which increases selectivity

for the cognate tRNA, possibly due to conformational changes within the aaRS for

proper positioning of the substrate (Guth & Francklyn, 2007; Yamane & Hopfield,

1977).

The maintenance of the genetic code relies on the correct pairing of amino acids

and cognate tRNAs. Due to their size and complexity, tRNAs offer sufficiently diverse

recognition elements to allow their specific selection by the corresponding aaRSs.

Selection of the proper amino acids from a pool of structurally related small molecules,

however, poses a more complex problem for aaRSs. For example, the small differences

in the potential binding energies of the aliphatic amino acids valine, isoleucine, and

leucine preclude a high level of specificity by the aaRSs and errors in aminoacylation

may occur (Table 1.2).

8 Class aaRS Edited non-cognate amino acid substrate

Class I LeuRS Ile, Met, norleucine, norvaline, homocysteine, γ-, δ-hydroxyisoleucine, γ-, δ-hydroxyleucine IleRS Ala, Cys, Thr, Val α-aminobutyrate, homocysteine, homoserine ValRS Ala, Cys, Thr, Ser, α-aminobutyrate, homocysteine MetRS Homocysteine

Class II LysRS II Homocysteine, homoserine, ornithine ProRS Ala, Cys, 4-hydroxyproline AlaRS Gly, Ser PheRS Ile, Tyr ThrRS Ser SerRS Thr, Cys, serine hydroxamate

Table 1.2. AaRSs known to possess editing activities and their non-cognate substrates.

In order to preserve the fidelity of protein synthesis, misactivated amino acids and non-cognate amino acid:tRNA pairs must be efficiently hydrolyzed to avoid infiltration of the genetic code by non-cognate substrates. As a result, some aaRSs have evolved a proof-reading mechanism to specifically recognize and hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing) (Figure 1.4;[(Hati et al., 2006; Jakubowski & Fersht, 1981; Jakubowski, 1990;

Karkhanis et al., 2006; Roy et al., 2004; Wong et al., 2003)]). Post-transfer editing of misacylated tRNAs is accomplished at a secondary site, known as the editing domain, which is appended to the catalytic cores of several aaRSs (Roy et al., 2004; Tukalo et al., 2005; Wong et al., 2003). Editing of misacylated tRNAs is thought to occur via a movement of the flexible tRNA CCA-end from the synthetic site to the separate editing

9 site. In some cases, when the appended editing domain is absent or inactive, a discrete

free-standing editing protein may be employed.

PPi tRNA

LeuRS + Nor + ATP LeuRS • Nor-AMP LeuRS • Nor-tRNA

Post-transfer Pre-transfer editing editing LeuRS + Nor + AMP

Figure 1.4. Schematic for the editing of non-cognate amino acid substrates by aaRSs. LeuRS significantly reduces the misincorporation of non-cognate amino acids during protein synthesis by hydrolyzing misactivated and misacylated norvaline (Nor) by pre- and post-transfer editing, respectively.

To overcome the difficulty of discriminating between chemically similar amino

acids, such as valine, isoleucine, and leucine, several aaRSs employ a double-sieve

editing mechanism (Figure 1.5; [(Fersht & Dingwall, 1979; Fersht, 1998; Nureki et al.,

1998)]). The first sieve excludes larger amino acids from entering the activation site,

allowing both the cognate and smaller amino acids to be activated. A second, fine

sieve located in the editing site is then responsible for hydrolyzing misacylated tRNAs,

while excluding the correctly aminoacylated tRNAs. As observed in the case of

isoleucyl-tRNA synthetase (IleRS), the activation efficiency of the non-cognate amino

acid valine is only approximately 200-fold less than the cognate isoleucine, creating a

potentially high level of valine misincorporation into the growing polypeptide chain

(Nureki et al., 1998; Schmidt & Schimmel, 1995).

10

Editing site - Hydrolyzes non-cognate amino acids Activation site - rejects amino acids larger than cognate tRNAIle

Ala-tRNAIle Gly-tRNAIle Phe Val-tRNAIle Tyr Ile-tRNAIle Ala Gly Val Ile Ile-tRNAIle

Figure 1.5. Schematic of the double-sieve editing mechanism of IleRS. Accurate protein synthesis requires editing of tRNAs misacylated with the non-cognate amino acid. To reduce the misincorporation of the incorrect amino acid in translation, IleRS utilizes a double-sieve editing mechanism to hydrolyze misactivated non-cognate amino acids and misacylated tRNAs. Amino acids larger than the cognate isoleucine (Ile) substrate are excluded from the first sieve located in the active site, permitting Ile and smaller amino acids to be activated with ATP and transferred to tRNAIle. In the second sieve, the editing site, tRNAIle misacylated with non-cognate amino acids smaller than Ile are hydrolyzed, while Ile-tRNAIle is excluded and released for protein synthesis. Adapted from Fersht, 1998.

11 The double-sieve editing mechanism substantially lowers the error rate to less than 1 in

3000, compatible with the overall level of fidelity observed for translation (Ibba & Soll,

1999; Nureki et al., 1998; Roy & Ibba, 2006). Many aaRSs, such as LeuRS (Chen et al., 2000; Lincecum, Jr. et al., 2003; Tukalo et al., 2005), ProRS (Wong et al., 2003), phenylalanyl- (Roy et al., 2004), valyl- (Fukai et al., 2000) alanyl- (Beebe et al., 2003), methionyl- (Gao et al., 1994; Jakubowski, 1996), and threonyl- (Dock-Bregeon et al.,

2000) tRNA synthetases, possess comparable editing mechanisms to minimize the

degeneracy of the genetic code by clearing misactivated amino acids and misacylated

tRNAs (Table 1.3).

Class aaRS cis-editing trans-editing domain factor Class I LeuRS CP1 - IleRS CP1 - ValRS CP1 - MetRS Active site? -

Class II LysRS II Acitve site? - ProRS INS1 Ybak, ProX AlaRS AlaX-like AlaX PheRS B3/B4 - ThrRS N2 ThrRS-ed SerRS Active site? -

Table 1.3. The cis- and trans-editing factors of aaRSs. See text for details. 1 INS: inserted editing domain found in bacterial species, such as E. coli; S. cerevisiae also possess an editing-like domain located at the N-terminus, which is not functional in editing; an inserted editing domain is absent in higher eukaryotes.

12 Editing of misacylated tRNA differs between class I and II aaRSs (Geslain &

Ribas, 2004). The common proof-reading domains found in class I IleRS, LeuRS, and

ValRS are known as connective peptide 1 (CP1), which is a globular domain appended

to the catalytic core of the respective aaRS (Figure 1.6; (Chen et al., 2000; Fukai et al.,

2000; Fukunaga et al., 2004). The CCA-end of the misacylated tRNA is repositioned from the catalytic site to the CP1 domain, located about 40 Å away, and allows for hydrolysis of the smaller, non-cognate amino acids in the editing site, while excluding the cognate amino acid (Tukalo et al., 2005). A conserved aspartic acid residue in the

CP1 editing domain is essential for the editing mechanism, interacting with the amino group of the non-cognate amino acid and positioning the substrate for hydrolysis of the ester bond (Lincecum, Jr. et al., 2003; Mursinna et al., 2004; Williams & Martinis,

2006).

Class II PheRS harbors an editing site, located on a subunit distinct from the active site, to discriminate cognate phenylalanine from the non-cognate amino acid tyrosine (Roy et al., 2004). The PheRS editing site discriminates and properly positions the non-cognate substrate for nucleophilic attack via a catalytic water molecule (Kotik-Kogan et al., 2005; Ling et al., 2007). Editing of misacylated tRNAs

by ThrRS and AlaRS, class II aaRSs, is thought to proceed by means of a conserved

histidine residue present in the appended editing domain, which extracts a proton in the

chemical step of the editing reaction (Dock-Bregeon et al., 2004).

13

Figure 1.6. Crystal structure of Pyrococcus horikoshii LeuRS. The catalytic body of P. horikoshii LeuRS (truncated from the C-terminus by 156 amino acids) and the separate editing domain (CP1) shown in cyan. From Fukunaga et al., 2004.

14 In some cases when an aaRS does not possess an editing activity of its own,

auxiliary protein factors are recruited to edit misacylated tRNAs (Table 1.3). Such is the case for the trans-acting factor YbaK, which specifically hydrolyzes Cys-tRNAPro

synthesized by ProRS (see section 1.4; (Ambrogelly et al., 2002; An & Musier-Forsyth,

2004; An & Musier-Forsyth, 2005). Other forms of deacylases are also found in

bacteria and eukaryotes. For example, D-Tyr-tRNA deacylase is responsible for

accelerating the hydrolysis of D-Tyr-tRNATyr, D-Asp-tRNAAsp, and D-Trp-tRNATrp

into free D-amino acid and tRNA, while rejecting the aa-tRNAaa substrates with an esterified L-amino acid (Lim et al., 2003; Yang et al., 2003). A paralog of

D-Tyr-tRNA deacylase has also been discovered in some archaeal species (Ferri-Fioni et al., 2006). Hydrolysis of the D-amino acid by D-Tyr-tRNA deacylase substantially lowers the misincorporation of D-amino acids into proteins (Calendar & Berg, 1967).

1.4. Bacterial aminoacyl-tRNA synthetase complexes in translation

Bacterial aaRSs are generally thought to function as stand-alone proteins to carry out the fundamental task of aminoacylation. However, a handful of binary complexes comprised of one aaRS and a second protein factor have been discovered in bacteria, with one early report suggesting the formation of a higher order multi-aaRS complex (Table 1.4; (Harris, 1987). These binary complexes are involved in a diverse range of functions, from editing of misacylated tRNAs to possible roles in metabolite biosynthesis.

15 aaRS (Class) Accessory protein Species Function

ProRS (II) YbaK H. influenza, E. coli Hydrolysis of Cys-tRNAPro AspRS (II) GatCAB amidotransferase T. thermophilus Conversion of Asp-tRNAAsn to Asn-tRNAAsn TrpRS (II) Nitric oxide synthase D. radiodurans Nitration of tryptophan, producing 4-nitro Trp

Table 1.4. AaRS complexes in bacteria. See text for details.

An elaborate network of protein-protein interactions is required for efficient

translation in all domains of life. The stable complex between ProRS and the free-

standing editing protein YbaK lends support to this notion. ProRS is known to

misacylate tRNAPro with non-cognate amino acids alanine and cysteine (Ambrogelly et al., 2002; An & Musier-Forsyth, 2004; An & Musier-Forsyth, 2005). Although most

ProRS enzymes have editing domains that efficiently deacylate Ala-tRNAPro, ProRS is unable to clear Cys-tRNAPro (An & Musier-Forsyth, 2004; Beuning & Musier-Forsyth,

2001; Ruan & Soll, 2005). To overcome this deficiency in bacteria, ProRS forms stable interactions with YbaK, a free-standing homologue of the ProRS Ala-tRNAPro

editing domain (An & Musier-Forsyth, 2004; An & Musier-Forsyth, 2005; Ruan &

Soll, 2005). YbaK, a general binding protein of tRNA, does not specifically recognize

tRNAPro, necessitating protein interactions with ProRS for correct substrate binding and

hydrolysis of Cys-tRNAPro, further guarding against the incorporation of non-cognate amino acids into the growing polypeptide chain. In competition studies, YbaK is unable to compete with EF-Tu for Cys-tRNAPro, suggesting that YbaK functions at a stage prior to product release from ProRS. Due to the inability of YbaK to specifically recognize tRNAPro and the inability to compete with EF-Tu for binding of the

16 mischarged species once released from ProRS, it is clear that complex formation

between YbaK and ProRS is essential for efficient hydrolysis of mischarged

Cys-tRNAPro.

While some mischarged aa-tRNAs are hydrolyzed by appended or discrete editing domains, protein synthesis in many bacterial species relies on the incorrect

coupling of certain amino acid and tRNA pairs. Such is the case for the synthesis of

Asn-tRNAAsn and Gln-tRNAGln, which proceeds via the specific mischarging of tRNAAsn and tRNAGln by aspartyl- and glutamyl-tRNA synthetases (AspRS and

GluRS), respectively (Figure 1.7; (Bailly et al., 2007; Feng et al., 2005; Schon et al.,

1988). The indirect synthesis of Asn-tRNAAsn requires a two-step reaction in which

first a non-discriminating AspRS mischarges tRNAAsn with aspartic acid (Becker &

Kern, 1998; Cardoso et al., 2006; Curnow et al., 1996; Min et al., 2003; Tumbula-

Hansen et al., 2002). The misacylated Asp-tRNAAsn is then bound by the GatCAB amidotransferase (AdT), which functions to convert the aspartic acid moiety to asparagine in the presence of an amide donor. Similarly, in bacteria Gln-tRNAGln is synthesized via an indirect pathway, which requires misacylation of tRNAGln by GluRS

and subsequent conversion to Gln-tRNAGln by the GatCAB Adt (Curnow et al., 1998;

Oshikane et al., 2006; Rocak et al., 2002; Salazar et al., 2001). The synthesis of

Asn-tRNAAsn and Gln-tRNAGln via indirect pathways is essential for those bacterial species whose genomes do not code for asparaginyl- or glutaminyl-tRNA synthetases

(AsnRS or GlnRS, respectively), providing the sole route for biosynthesis of these amino acid:tRNA pairs.

17

A. Asn Direct pathway

Asn-tRNAAsn AsnRS

Asn Asn tRNA specific tRNA transamidase Non-discriminating (Asp-AdT) AspRS Asp-tRNAAsn

Asp + ATP Asn + ATP Asp Indirect pathway

B. Gln Direct pathway

Gln-tRNAGln GlnRS

Gln Gln tRNA specific tRNA transamidase Non-discriminating (Glu-AdT) GluRS Asp-tRNAGln

Glu + ATP Gln + ATP Glu

Indirect pathway

Figure 1.7. Two-step pathways for the formation of Asn-tRNAAsn and Gln- tRNAGln. Pathway for the direct and indirect (two-step amidotransferase pathway) synthesis of Asn-tRNAAsn (A) and Gln-tRNAGln (B). Adapted from Francklyn et al., 2001.

18 The rate-limiting step in this tRNA-dependent reaction is amide transfer prior to

the formation of Asn-tRNAAsn (Bailly et al., 2007). Recently, it was discovered that

the bacterial AspRS stably associates with AdT in the presence of tRNAAsn, forming

the transamidosome complex. Within the context of the transamidosome, the rate of

Asp-tRNAAsn conversion to Asn-tRNAAsn was significantly enhanced, as compared to

free AdT. This suggests that the transfer of Asp-tRNAAsn from AspRS to AdT requires stable complex formation between the two proteins for efficient conversion. Although it has not been observed in vitro, a similar complex between the non-discriminating

GluRS and AdT has also been suggested to occur in the cell (Bailly et al., 2007;

Oshikane et al., 2006). The transamidosome may be necessary not only to enhance the synthesis of Asn-tRNAAsn, but may also channel the mischarged aa-tRNA intermediate directly from AspRS to the AdT. As these two proteins execute consecutive functions, protein-protein interactions may be important to facilitate the direct transfer of the vital

Asp-tRNAAsn intermediate from AspRS to the AdT, preventing spontaneous hydrolysis in the cytoplasm.

In many bacteria, multiple copies of aaRSs are often encoded in the same genome (Ataide et al., 2005; Ataide & Ibba, 2006; Brown et al., 2003; Gentry et al.,

2003; Henkin et al., 1992; Levengood et al., 2004; Putzer et al., 1992; Salazar et al.,

2003). One example is Deinococcus radiodurans, which harbors two genes encoding

tryptophanyl-tRNA synthetase (TrpRS I and TrpRS II; (Buddha et al., 2004a; Buddha

et al., 2004b). Although the two TrpRSs contain the conserved amino acid residues

responsible for aminoacylation of tRNATrp with tryptophan (Trp), TrpRS II is unusual

in that it is structurally more similar to human TrpRS and is induced in response to

19 damage caused by radiation (Buddha et al., 2004a). In addition, TrpRS II contains an

N-terminal extension similar to other proteins involved in stress responses in bacteria

and has been shown to associate with nitric oxide synthase (NOS) (Buddha et al.,

2004a; Buddha et al., 2004b). In mammals, nitric oxide is synthesized from the

oxidation of arginine (Arg), and functions in nerve signal transmission, vasodilation,

and the immune system (Kers et al., 2004). Some bacterial species harbor truncated

homologues of the mammalian NOS, which have been identified to perform novel

functions in bacteria. In complex with TrpRS II and Trp-tRNATrp, the bacterial NOS

catalyzes the regiospecific nitration of Trp from Arg at the 4-position, producing

4-nitro Trp (Buddha et al., 2004b). Complex formation between TrpRS II and NOS

favored the activities of both associated enzymes. TrpRS II increased NOS solubility

and affinity for its Arg substrate, facilitating the specific nitration of Trp from Arg

(Buddha et al., 2004b; Buddha & Crane, 2005b). TrpRS II may also supply NOS with

adenylated-Trp for subsequent nitration or nitrosylation, as Trp nitration does not occur

with free Trp in vitro (Buddha et al., 2004a; Buddha et al., 2004b).

Both TrpRS I and TrpRS II are able to aminoacylate tRNATrp with Trp,

although the efficiency of TrpRS II is about 5-fold lower than TrpRS I (Buddha et al.,

2004a). In addition to the canonical Trp substrate, TrpRS II has been observed to charge tRNATrp with 4-nitro Trp in the presence of ATP with nearly the same aminoacylation efficiency as with Trp (Buddha & Crane, 2005a). Although the role of

TrpRS II and 4-nitro Trp is largely unknown, it may function outside of protein synthesis as D. radiodurans encodes two TrpRSs, with TrpRS II expression only

induced in response to specific stimuli such as radiation damage. 4-nitro Trp may play

20 a role in the synthesis of other metabolites or may perhaps function in a role similar to

that of charged tRNA in cell envelope remodeling, where the aa-tRNA functions outside of protein synthesis to provide amino acids required for cell membrane biosynthesis (Ibba & Soll, 2004; Kers et al., 2004; Roy & Ibba, 2008).

A tRNA-binding protein (Trbp111) discovered in bacteria is also an attractive candidate for protein-protein interactions with aaRSs. Trbp111 has been observed to bind the outer, convex side of the tRNA molecule, while aaRSs bind the inner, concave portion (Morales et al., 1999; Swairjo et al., 2000). Biochemical studies have suggested that Trbp111 forms a ternary complex with IleRS, mediated via interactions with disparate portions of the tRNA (Nomanbhoy et al., 2001). It is likely that other higher order protein complexes containing aaRSs are more widespread in the cell and are awaiting discovery. A large-scale protein-protein interaction map of Helicobacter

pylori lends support to this notion, cataloging an extensive list of protein complexes

(Rain et al., 2001). These genetic screens provide an excellent approach to begin to

unravel the intricate web of protein-protein interactions that occur in vivo and may be

used to enhance our understanding of functional associations based on the involvement

of at least one protein factor from known cellular pathways.

1.5. Aminoacyl-tRNA synthetase complexes in Saccharomyces cerevisiae

A variety of multi-protein complexes containing aaRSs exists throughout all

three domains of life. In eukaryotes these complexes tend to be larger than those

discovered in bacteria and also perform a wider range of functions from enhanced

aminoacylation to non-canonical functions beyond translation (Table 1.5). In

21 Saccharomyces cerevisiae, GluRS and MetRS stably interact with aminoacyl-tRNA

synthetase cofactor I (Arc1p), a non-synthetase accessory protein (Galani et al., 2001;

Karanasios et al., 2007; Simos et al., 1996; Simos et al., 1998). Protein-protein interactions are mediated through the N-terminal domains of each associated component, as observed from biochemical studies and from modeling of two binary complexes of Arc1p with each aaRS (Deinert et al., 2001; Simader et al., 2006).

Discrete residues of Arc1p have been identified as critical for binding GluRS and

MetRS, mediating ternary complex formation (Galani et al., 2001; Karanasios et al.,

2007). Arc1p harbors an EMAPII-like domain at its C-terminus, containing an oligonucleotide binding (OB) domain, and a positively charged central domain, which enables Arc1p to act as a general tRNA binding factor (Simos et al., 1996; Simos et al.,

1998). As part of the ternary complex, Arc1p preferentially facilitates the binding of the cognate tRNA substrates (tRNAGlu and tRNAMet) to the associated aaRSs, thus increasing the catalytic efficiencies of both GluRS and MetRS (Galani et al., 2001;

Simader et al., 2006; Simos et al., 1998).

22 m ; ; g g as ; n on) n s ond i Glu l u y g opl ng; t e n l y be c nneli eli eli a nu h hanne he c nd tRNA c hann hann a the s c t to t c Ly m unctions Me ns o tRNA NA r NA A f SerRS tR s aa-tRNA c to RNA aa- on during sporulati i t ; t f tRN aaRS f y f a Ser it ; aa-tR ; aa- o il n n y rm b ease; aaRS o t ng o l i ated protei o a tio i f e t tion ni a i at l tRNA a f agi l z f f y y n s on of c anonical i a o c c ei assoc us ocali rosine l lencing f l i ; pack inoa y noa c i t r y o product r x s l nding nding m a non-

n e m p b f * ed prot lul As tio o m ( c a el n (*di n ional ed a l ol t ed a riction se c tion t tr a l n c nc s tRNA anc as now now o l res sub n h trans c enhan hanced bi hanced bi a ha p- a s. r r l En En Unk En T En Vi As Fun Unk

r detai

o s s te ens visiae visiae visiae ens ens ens ens i ie e e e p r r r da c a api api api api e e e c c c . . . H. s S H. s H. s S H. s S H. s Chor See text f Spe 1 P

oes.

y y s) DH, NSA p38 or g P , p43 n( a A and ei ess α , G α p α 21p 1 a 1 c 1 1 r4 3 s in eukar c n 1 e EF- K Arc prot HIV-1 G Pex EF- L A lex I), p18, p38 ( p I), EF-

( S m R

, Ile I) ( ,

II) I) I) ( ( RS co ( a ) S R pRS (I, II), MetRS u l ass II) As LeuRS I), ArgRS l (I, II) ( ( , , c ) II) I), G II) II) II) II) I I) ( ( ( ( ( ( ( ( S S S S R R R R GluProRS GlnRS AspRS pRS rRS s s s uProRS l y y y y Table 1.5. A aaRS ( MetRS L ValRS L G L T As Ser

23 Bacterial Trbp111 and human p43 (an auxiliary protein of the mammalian

MSC, see section 1.6) both harbor tRNA binding domains similar to that found in the

C-terminus of Arc1p and can in fact functionally replace Arc1p to complement a

synthetic lethal arc1- los1- strain of S. cerevisiae (Deinert et al., 2001; Golinelli-Cohen et al., 2004; Quevillon et al., 1997; Simos et al., 1996; Swairjo et al., 2000). Based on models of Trbp111, Arc1p has been suggested to interact with the outer portion of the tRNA substrate, which is also in agreement with biochemical studies implicating the tRNA D- and TΨC-loops as part of the binding site for Arc1p (Simos et al., 1996;

Swairjo et al., 2000). Both GluRS and MetRS are class I aaRSs, known to interact with the inner L portion of the tRNA molecule, which may permit simultaneous associations of Arc1p and aaRSs with their tRNA substrates in complex (Sekine et al., 2001; Senger et al., 1995).

Arc1p in complex with GluRS and MetRS has been observed to preferentially facilitate the binding of tRNAGlu and tRNAMet to each aaRS, possibly by promoting

structural rearrangements required for efficient binding of the tRNA substrate and

aminoacylation (Galani et al., 2001; Golinelli-Cohen & Mirande, 2007; Simader et al.,

2006; Simos et al., 1998). Although unbound GluRS is able to bind its cognate

tRNAGlu with moderate affinity, tRNA binding is greatly influenced by the presence of

Glu Arc1p. In complex, the apparent KD for tRNA is reduced about 120-fold to 30 nM

(Graindorge et al., 2005). Steady-state aminoacylation kinetic parameters could not be determined, however, since in vitro transcribed tRNAGlu proved a poor substrate for aminoacylation (Graindorge et al., 2005). In the case of MetRS, the kcat for tRNA aminoacylation increased almost five-fold in the presence of Arc1p as compared to

24 MetRS alone (Simos et al., 1996). Arc1p facilitated the binding of tRNAMet to MetRS

Met by significantly decreasing (about 100-fold) the KM of tRNA , as compared to the

Phe aaRS alone (KM ~10 µM). Further, tRNA was unable to compete for the interaction with MetRS, indicating that MetRS is responsible for specific interactions with the tRNAMet substrate.

Beyond its role in enhanced binding of cognate tRNAs to MetRS and GluRS, the Arc1p complex also regulates the subcellular localization of each associated component (Galani et al., 2001; Golinelli-Cohen & Mirande, 2007). Arc1p is exclusively found in the cytoplasm due to export by Xpo1p (Galani et al., 2001; Galani et al., 2005). When associated with Arc1p, both GluRS and MetRS localize to the cytoplasm, while disruption of the complex due to N-terminal truncations of each protein allows entrance of the individual aaRSs into the nucleus (Galani et al., 2001;

Galani et al., 2005; Karanasios et al., 2007). Arc1p may also interact indirectly with

Los1p (a nuclear pore-associated protein involved in tRNA export), as discovered from synthetic lethal screens using a disrupted los1- strain, indicating the involvement of

Arc1p in tRNA export from the nucleus (Simos et al., 1996; Simos et al., 1998).

Translation factor EF-1α was also discovered in a suppressor screen of a synthetically

lethal los1- strain, suggesting that EF-1α may usher newly exported tRNA from the

nucleus to the corresponding aaRSs (Grosshans et al., 2000b; Grosshans et al., 2000a;

McGuire & Mangroo, 2007; Sarkar et al., 1999). Taken together, these data reveal how

Arc1p coordinates events between protein synthesis and tRNA export in S. cerevisiae.

In addition to the Arc1p:GluRS:MetRS complex, S. cerevisiae also harbors a

complex between seryl-tRNA synthetase (SerRS) and Pex21p, a protein involved in 25 peroxisome biosynthesis. First identified by yeast two-hybrid analysis, pull-down

experiments indicated that Pex21p and SerRS associate in cell-free extracts, suggesting

complex formation in vivo (Rocak et al., 2002). The C-terminus of SerRS, which is not

essential for cellular viability or aminoacylation, mediates protein-protein interactions

with Pex21p (Godinic et al., 2007; Mocibob & Weygand-Durasevic, 2008). In

complex, Pex21p promoted the binding of tRNASer to SerRS, slightly enhancing aminoacylation (Godinic et al., 2007; Rocak et al., 2002). Pex21p may induce conformational changes in SerRS to enhance the binding of cognate tRNA, playing a similar role to Arc1p in complex with GluRS and MetRS (Godinic et al., 2007; Rocak

et al., 2002; Simader et al., 2006; Simos et al., 1998).

Peroxisomes, which are involved in the metabolism of fatty acids and other

metabolites, function as part of the secretory pathway to post-translationally import

peroxisomal enzymes across the peroxisome membrane. Although the functional

consequences of complex formation on the activity of Pex21p remain to be discovered,

the SerRS:Pex21p complex provides a connection between translation and peroxisome

biosynthesis. SerRS is also involved in the synthesis of diadenosine oligophosphates,

which may act as a signal of oxidative stress (Lopez-Huertas et al., 2000). Peroxisome

biosynthesis genes are also known to be induced by hydrogen peroxide and other

stressors to the cell, suggesting that complex formation between SerRS and Pex21p

may contribute to coordination of stress signaling networks in S. cerevisiae (Belrhali et

al., 1995; Godinic et al., 2007; Lee et al., 1983).

A connection between tyrosyl-tRNA synthetase (TyrRS) and a protein involved

in cell wall biosynthesis (Knr4p) has also been identified in S. cerevisiae from DNA

26 microarray, yeast two-hybrid, and pull-down experiments (Dagkessamanskaia et al.,

2001; Ivakhno & Kornelyuk, 2005). DNA microarray analyses demonstrated that

TyrRS gene expression during sporulation correlated well with the expression of

Knr4p, as well as other genes that participate in cell wall assembly (Ivakhno &

Kornelyuk, 2005). Disruption of the gene encoding Knr4p reduced glucan synthase activity and resulted in decreased spore formation, which was associated with a concomitant activation of TyrRS during sporulation (Chu et al., 1998). Although the biological function of this cellular association is largely unknown, dityrosine is an essential component of the outermost layer of the spore wall, suggesting a collaborative effect between TyrRS and Knr4p to promote dityrosine formation during sporulation in

S. cerevisiae (Briza et al., 1986; Coluccio et al., 2004).

1.6. Mammalian multi-aminoacyl-tRNA complexes in translation

In mammalian cells, a large 1.4 MDa multi-aaRS complex exists, composed of nine aaRS activities (LysRS, LeuRS, GluProRS, IleRS, MetRS, GlnRS, ArgRS, and

AspRS) (Bandyopadhyay & Deutscher, 1971; Kerjan et al., 1994; Quevillon et al.,

1999; Robinson et al., 2000). Three non-aaRS factors p38, p43, and p18 complete the complex, playing roles in tRNA binding and complex stability/assembly (Figure 1.8).

The MSC is composed of a mixture of class I and class II aaRSs, indicating that the distinction between the complexed and free forms does not solely reside in the structural architectures of their active sites nor the amino acids that are activated and attached to their corresponding tRNA molecules. It has been observed that the aaRSs responsible for coupling charged amino acids and hydrophobic, non-aromatic amino

27 acids to tRNA molecules are all present within the complex, while those aminoacylating the smallest and largest amino acids are absent (Wolfson & Knight,

2005). It is unknown why certain aaRSs unite into a larger complex while others exist as free-standing proteins. It is possible, however, that several other, or perhaps even all, aaRSs associate with the complex more transiently in the cell and the isolated MSC composition may reflect experimental limitations when co-purifying macromolecular complexes.

Although the composition of the proteins stably associated in the mammalian

MSC was deciphered nearly thirty years ago, the exact structural arrangement and assembly of the complex remain undefined. Through biochemical, genetic, and cryo- electron microscopic analyses, however, a clearer picture of the MSC has been captured

(Kim et al., 2000; Norcum, 1989; Norcum, 1999; Quevillon et al., 1999; Robinson et al., 2000; Wolfson & Knight, 2005; Han et al., 2003). The MSC particle is compact, as visualized by electron and immunoelectron microscopy, and assembles into a V-shaped complex, possibly via the association of three subcomplexes (Norcum, 1989; Norcum

& Warrington, 1998; Norcum, 1999). Localized in one arm are AspRS, MetRS, and

GlnRS, while on the other arm are LysRS and ArgRS. The bifunctional GluProRS,

IleRS, and LeuRS are found at the base of the V-shaped macromolecule, and the polypeptides connect the three subdomains, forming the complete mammalian MSC

(Wolfe et al., 2005).

28

A. B.

p38 D-RS D KK-RS M-RS Q R-RS 90º p43

C.

Figure 1.8. Schematic diagram of the mammalian multi-aaRS complex. Based on chemical cross-linking and cryo-electron microscopy data, the mammalian MSC composed of nine aaRS activities and three auxiliary proteins is organized into a V-shaped particle containing three segments (two arms and a base). The precise location of p18 is unknown. Adapted from Norcum et al., 1998 and Wolfe et al., 2005.

29 The assembly of the MSC is thought to be highly structured and interdependent on other binding events within the complex. Lending support to the notion of ordered

assembly, NaSCN or high concentrations of NaCl result in the release of LysRS and

AspRS from the complex, respectively, suggesting that these aaRSs are located near the

periphery (Agou & Mirande, 1997; Cirakoglu et al., 1985; Norcum, 1991). MetRS,

GlnRS, and ArgRS are also relatively easy to remove from the MSC and have been

found as free forms in the cell (Han et al., 2006a; Han et al., 2006b; Ko et al., 2001).

Extensive pair-wise yeast two-hybrid screening of each aaRS associated in the MSC

was performed, providing insight into all 64 combinations of protein-protein

interactions within the complex (Rho et al., 1999). Protein-protein interactions within

the mammalian MSC may be mediated through appended domains found in the

associated aaRSs, as has been observed for MetRS, LeuRS, and ArgRS (Guigou et al.,

2004; Ling et al., 2005; Mirande et al., 1982b; Mirande et al., 1992; Robinson et al.,

2000; Vellekamp & Deutscher, 1987). Conversely, several aaRSs may associate with

the MSC through their catalytic domains (LysRS, AspRS, and GlnRS), since deletion

of the appended extensions did not abolish interactions with the MSC (Agou &

Mirande, 1997; Francin et al., 2002; Kim et al., 2000; Robinson et al., 2000). The

interdependence of binding events was further evidenced by the fact that the binding of

GlnRS to p38 was significantly enhanced in the presence of p43 and ArgRS, indicating

that perhaps a discreet quaternary complex is required for the ordered formation of the

MSC (Robinson et al., 2000).

In addition to the nine aaRS activities associated within the MSC, three

auxiliary polypeptides (p38, p43, and p18) complete the complex, lending stability to

30 the complex and promoting the binding of tRNA. The auxiliary protein p38 has been

observed to mediate protein-protein interactions, acting as an essential scaffolding protein of the mammalian MSC (Ahn et al., 2003; Kim et al., 2002; Robinson et al.,

2000). p38 interacts with nearly all other proteins associated in the MSC over a range of binding affinities between 0.3 nM - 5 µM, emphasizing its role as an indispensable core protein important for assembly and stability of the complex (Ahn et al., 2003; Kim et al., 2002; Quevillon et al., 1999; Robinson et al., 2000; Wolfe et al., 2005).

Although the molecular mechanisms are not well-defined, p38 has also been implicated in roles outside of the MSC. For example, p38 has been observed to associate in the nucleus with FUSE-binding protein (a transcriptional activator of c-myc), resulting in ubiquitination of FUSE-binding protein and subsequent down- regulation of c-myc (Kim et al., 2002; Kim et al., 2003). p38 has also been shown to associate with and act as a substrate of Parkin (a protein involved in ubiquitination), which results in the degradation of p38 (Corti et al., 2003; Ko et al., 2005).

Autosomal-recessive juvenile parkinsonism is caused by loss-of-function mutations in the gene encoding Parkin, which lead to degeneration of dopaminergic neurons via accumulation of toxic proteins. It has been observed that loss of Parkin function results in the accumulation of non-ubiquitinated p38, which leads to the formation of aggresome-like inclusions. Although the mechanism remains largely unknown, this accumulation of p38 may contribute to dopaminergic neurodegeneration, providing a link between a protein normally associated in the mammalian MSC and the neurodegenerative disorder Parkinson’s Disease.

31 The p38 polypeptide associates with a second auxiliary protein, p43, which is

also an integral part of the MSC (Wolfe et al., 2003). The C-terminus of p43 harbors

an EMAPII-like domain (endothelial monocyte-activating polypeptide II), which

confers a general tRNA binding property (Deinert et al., 2001; Quevillon et al., 1997;

Simos et al., 1996; Swairjo et al., 2000). This domain is homologous to the yeast

Arc1p protein that facilitates the binding of the cognate tRNA substrates to MetRS and

GluRS (Golinelli-Cohen & Mirande, 2007). The EMAPII-like domain, which may be removed from the full-length p43 following apoptotic cleavage, is involved in altering endothelial functions as a proinflammatory cytokine (Behrensdorf et al., 2000; Faisal et

al., 2007; Shalak et al., 2001). The p43 EMAPII domain has also been shown to

upregulate proinflammatory genes and increase chemotactic migration of

polymorphonuclear leukocytes upon cleavage and removal from the MSC (Behrensdorf

et al., 2000; Park et al., 2002). The multi-functionality of these proteins involved in the mammalian MSC emphasizes the interconnection between translation and cellular processes outside of protein synthesis.

Although the role of the third auxiliary factor p18 remains largely unknown, p18 shares homology with the N-terminus of human ValRS, which is responsible for mediating complex formation with EF-1H (Park et al., 2005a; Quevillon & Mirande,

1996; Sang et al., 2002). EF-1α has also been observed to associate with LysRS, a member of the mammalian MSC, suggesting that EF-1α may transiently associate with the macromolecular complex to provide an efficient means to shuttle aa-tRNA directly from the MSC to the ribosome during translation (Guzzo & Yang, 2008; Sang et al.,

2002). AaRSs have also been observed to co-migrate with polysomes (Popenko et al., 32 1994). Whether aaRSs form specific complexes with polysomes or rather associate

transiently remains to be determined.

Increased protein stability or restriction of the associated aaRSs to the cytoplasm, as was observed for the yeast Arc1p:GluRS:MetRS complex, may entice aaRSs to associate into the higher order MSC complex (Galani et al., 2001). The

functional association of nine aaRS activities has also been suggested to provide a

sequestered pool of aa-tRNAs specifically for utilization in protein synthesis

(Negrutskii & Deutscher, 1991; Negrutskii & Deutscher, 1992). Radiolabeled

aa-tRNA, introduced into permeabilized CHO cells, were unable to be used as

substrates for protein synthesis, while radiolabeled amino acids were efficiently

incorporated during translation (Negrutskii & Deutscher, 1992). This suggests that

endogenously aminoacylated tRNAs, synthesized within the MSC, are required for

efficient protein synthesis and that the pools of aa-tRNAs within the cell do not mix freely. This is supported by studies of ArgRS, which exists as both complexed (full- length) and free (truncated at its N-terminus) forms, translated from alternative start codons (Sivaram & Deutscher, 1990; Zhang et al., 2006). Recently, it was observed that the pool of aa-tRNAs synthesized by ArgRS complexed with the MSC was preferentially utilized as a substrate for protein synthesis in vivo (Kyriacou &

Deutscher, 2008). Although the precise molecular mechanism remains to be

deciphered, the work by Deutscher and colleagues lends support to the notion that the

mammalian MSC is intimately involved in protein synthesis by providing the

elongating ribosome with amino acid substrates synthesized within the MSC

(Negrutskii & Deutscher, 1991; Negrutskii et al., 1999). A larger complex of aaRSs

33 was also discovered in the nucleus, possibly functioning in a role similar to the

cytoplasmic MSC counterpart via association with nuclear pore-associated EF-1α or to

promote nuclear tRNA export (Ko et al., 2001; Nathanson & Deutscher, 2000; Popenko

et al., 1994). The mammalian MSC has been implicated in a variety of functions

including promotion of protein stability and coordination of events between aa-tRNA

synthesis and delivery to the ribosome during translation elongation. In light of the fact

that multi-aaRS complexes in all three domains of life remain largely undefined, further

studies are now necessary to deconvolute the biological role of MSCs and provide new

insight into their possible functions in translation.

1.7. AaRS complexes in Homo sapiens

Apart from the primary role of coupling cognate amino acid and tRNA pairs, it

has recently become increasingly evident that aaRSs are not limited to protein synthesis

(Table 1.6; (Greenberg et al., 2007; Herbert et al., 1988; Howard et al., 2002; Kise et

al., 2004; Ko et al., 2001; Lee et al., 2004; Park et al., 2005a; Paukstelis et al., 2008;

Rho et al., 2002; Romby et al., 1996; Torres-Larios et al., 2002)]). In fact, the MSC may act as a repository for aaRSs, where in response to cellular changes, aaRSs are

subsequently liberated from the complex to participate in non-canonical tasks beyond

translation (Ray et al., 2007; Park et al., 2005b; Lee et al., 2004). The bifunctional

GluProRS, which harbors two different aaRS catalytic activities separated by three

tandem linker domains, lends support to this notion by participating in the translational

silencing of ceruloplasmin (Cp; a protein linked to the inflammatory response) (Fox et

al., 2000; Ray et al., 2007; Rho et al., 1998; Sampath et al., 2004).

34 ) ) s s l l l l e e c

l ia l s e Ly h t o d ndothelial c e en tRNA

s ts e g of es t g in y ar g l l t g e g g a oc o o (target l r r in t t e ( ck uk c kin cin cin ro n n ne n s. i i t e i n l l l o o rRNA to ki pa l k p p

le r i ; o s s l c l c to t o f s cy of s y i n n l l con n cy c s c o o ry a o ona ona r detai ti i i i tr tr o c n na mbly to n ic t t t o c ti o e n io i o a in in t t s a t i e rip rip rip t I I a a o pop c c c l l g as p p s s s s s a os o i - u u i n ammat em g t l an a an an an h unct n r r r r r See text f Viral Inf Angi An A T T Gro C T Gro T T F n. iae s s s s s s s s s io t vis sa ies pien pien pien pien pien pien pien pien pien li li re as a a a a a a a a a ec s s s s s s s s s ansla co co ce r H. H. H. Sp H. H. H. H. N. cr H. E. S. H. E. nd t e o n a r bey r r r a a s i i a a a l l l S m m m m m u u l l ndr ndr llu on s i o o as as as el el as a memb l u h ce aaR p pl pl pl e f ac ac l o sm ra cat oc cleus toplas to to to tr tr t t i uc u x x x la o es o Mitoch Cy M Cyt Cy Cy E N E N P Cy E L ti i

) ) I ss (I, I la cal activ ) ) ) i I) I I I) II I) II) II) I) II) I) I II) ( ( n ( ( ( ( ( ( ( RS ( ( ( (C o S S s S s S S S S S no R a R R R R R rRS u r c s s sR nR p aR y y y e n- aaR L L Ty Tr Gl MetRS GluPr T HisRS Al Ly L Thr o a a i y r er a main k ct o Eu D Ba Table 1.6. N

35 Although generally associated with the MSC, GluProRS is phosphorylated in response to IFN-γ by an unknown mechanism, departs from the MSC, and is involved in mRNA silencing of Cp via the IFN-gamma activated inhibitor of translation (GAIT) complex composed of GluProRS, NSAP1, glyceraldehyde 3-phosphate dehydrogenase

(GAPDH), and ribosomal protein L13a (Figure 1.9; [(Agou & Mirande, 1997;

Cirakoglu et al., 1985; Norcum, 1991)]). Within the context of this heterotetrameric complex, translation is inhibited by the binding of the GluProRS linker region to a regulatory stem-loop GAIT mRNA element located within the 3’ untranslated region

(UTR) of Cp.

First, a pre-GAIT complex of GluProRS and NSAP1 is formed that is unable to bind the 3’ UTR region of Cp and silence translation, possibly due to the inhibitory effects of NSAP1 (Jia et al., 2008). The association of L13a and GAPDH complete the heterotetrameric GAIT complex, inducing a conformational change that abrogates the inhibitory effects of NSAP1. The GAIT complex is then able to bind the GAIT mRNA element of Cp, mediated by the GluProRS WHEP domains, resulting in translation inhibition in response to IFN-γ (Kapasi et al., 2007; Mazumder et al., 2003a;

Mazumder et al., 2003b; Sampath et al., 2004). Although the precise molecular mechanism of translational silencing via the GAIT complex is not well-defined, GAIT mRNA elements are found in more than 30 human mRNAs, suggesting a coordinated regulation of the inflammatory response via a complex involving the multi-functional

GluProRS (Mazumder et al., 2003a; Mazumder et al., 2003b; Sampath et al., 2004).

36

Figure 1.9. Schematic of translational silencing by the GAIT complex. GluProRS is phosphorylated and removed from the mammalian multi-synthetase complex. GAPDH, phosphorylated L13a, and NSAP associate with GluProRS, forming the GAIT complex capable of binding the GAIT mRNA element and silencing the associated gene product. From Ray et al., 2007.

37 AaRSs have also been implicated in a number of functions separate from

translation, including involvement in human immunodeficiency virus type 1 (HIV-1)

viral assembly. Human HIV-1, an RNA retrovirus, enters the target cell and converts

its RNA genome into DNA using a virally encoded reverse transcriptase (Dobard et al.,

2007; McCulley & Morrow, 2007; Chan et al., 1999). Initiation of reverse

transcription requires the specific packaging of tRNALys into the HIV-1 virions, which functions as a primer for reverse transcriptase by binding near the 5’ end of the HIV-1 genomic RNA (Figure 1.10; [(Barat et al., 1989; Kleiman et al., 2004; Levin et al.,

2005; McCulley & Morrow, 2007)]). Following minus strand transfer and reverse transcription of the minus strand DNA, the plus strand DNA is synthesized and the double-stranded DNA copy is translocated into the nucleus of the target cell where it is integrated into the host’s chromosomal DNA (Liu et al., 2005; Zeng et al., 2007).

Human LysRS is also selectively packaged into the HIV-1 virion, independent of tRNALys incorporation, which is evidenced by the fact that LysRS mutants unable to

bind tRNA retain the ability to be packaged (Javanbakht et al., 2003). Incorporation of

LysRS into the virion is accomplished via specific interactions with HIV-1 Gag (a

dimeric protein involved in assembly of viral particles), which interacts stably with a

KD of about 0.3 µM (Guo et al., 2005; Javanbakht et al., 2003; Kovaleski et al., 2006).

Although the mechanism remains largely undefined, LysRS is believed to contribute positively to the selective incorporation of tRNALys. Lending support to this notion was

the discovery that inhibition of LysRS synthesis resulted in reduced tRNALys packaging

and annealing to the HIV-1 viral RNA (Guo et al., 2003).

38

1. tRNALys annealing PBSPBS HIV-1 RNA genome tRNALys

2. Reverse transcription PBSPBS HIV-1 RNA genome tRNALys (- strand) DNA

PBSPBS HIV-1 RNA genome 3. Minus strand transfer tRNALys

4. Elongation of (-) strand DNA; PBSPBS HIV-1 RNA genome degradation of RNA via RT tRNALys RNase H activity (- strand) DNA

5. Synthesis of (+) PBSPBS HIV-1 ds DNA (+) strand DNA (-) PBS

Figure 1.10. Schematic of the events in HIV-1 reverse transcription. Step 1. Reverse transcription is initiated by the binding of tRNALys to the primer binding site (PBS) of the HIV-1 genomic RNA. Step 2. Virally-encoded reverse transcriptase (RT) extends the minus (-) strand DNA. Step 3. The minus strand is transferred to and binds the 3’ end of the RNA, acting as a primer for reverse transcription of the RNA genome. Step 4. Elongation of (-) strand DNA and degradation of the RNA via RNase activity of RT (hashed lines). Step 5. Synthesis of plus (+) strand DNA, resulting in double- stranded (ds) DNA copy of the HIV-1 genome. Adapted from Levin et al., 2005.

39 The interaction interface between LysRS, a dimeric class II aaRS, and Gag has

been mapped to the dimerization domains of each protein, although the precise

stoichiometry and multimeric form of each enzyme remain largely unknown

(Javanbakht et al., 2003; Kovaleski et al., 2006). It has been postulated that newly synthesized LysRS associates with Gag rapidly prior to joining the MSC (Halwani et al., 2004). This demonstrates that LysRS, which is an essential part of the mammalian

MSC required for efficient protein synthesis, has been recruited from its primary role in

translation to take part in the auxiliary function of HIV-1 viral assembly and packaging

of its cognate tRNA.

1.8. Association of aaRSs with the translation factor EF-1α

In eukaryotes, aaRSs are also known to associate into higher order complexes with the translation factor EF-1α (see Table 1.5). Translation elongation requires the

correct synthesis of aa-tRNA by aaRSs and subsequent delivery to the ribosome by

EF-1α. In human cells, the GTPase EF-1α associates with the guanine nucleotide

recycling machinery, EF-1βδγ, to form the larger elongation factor 1 H (EF-1H)

complex. Upon codon-anticodon recognition on the ribosome, GTP is hydrolyzed,

releasing the GDP-bound form of EF-1α. EF-1βδγ then functions to recycle GDP to

GTP, permitting EF-1α to pursue another round of tRNA selection. A stable complex

between human ValRS and EF-1H provides a link between these essential steps in

translation (Bec et al., 1994; Minella et al., 1998; Motorin et al., 1988; Negrutskii et

al., 1999; Venema et al., 1991). The N-terminal appended domain of ValRS was

40 identified to mediate protein-protein interactions, as it strongly associates with the

EF-1δ subunit of EF-1H (Negrutskii et al., 1999). Although the stoichiometry of the

EF-1H:ValRS complex is debated, the presence of excess EF-1α and GTP enhanced

Val the catalytic efficiency of ValRS almost two-fold, while the KM for tRNA remained unaffected (Negrutskii et al., 1999). This increase in the catalytic efficiency was not observed in the presence of EF-1α·GDP or bacterial EF-Tu·GTP, indicating that the increased levels of Val-tRNAVal were due to improved catalysis by ValRS, and not the protective effects of EF-1α.

It has been suggested that aminoacylation by class I aaRSs is rate-limited by aa-tRNA release, while class II enzymes are rate-limited by a step prior to product release (Zhang et al., 2006). These distinctions between class I and class II aaRSs may compel an association of translation elongation factors with class I aaRSs for efficient aa-tRNA release while class II aaRSs may not necessarily require stable interactions with EF-1α for product release (Zhang et al., 2006). The EF-1α:ValRS complex correlates well with the ability of the elongation factor to form complexes with and enhance the rate of aminoacylation by class I aaRSs (Kern & Gangloff, 1981; Zhang et al., 2006). EF-1α has also been observed to associate with a class II aaRS, human

AspRS, which stimulated the rate-limiting step of Asp-tRNAAsp release (Reed & Yang,

1994; Reed et al., 1994). More recently, EF-1α has been suggested to interact with

class II human LysRS, slightly enhancing aminoacylation by LysRS (Guzzo & Yang,

2008). An interaction between human TrpRS and EF-1α has also been proposed,

although stable complex formation has not yet been confirmed (Yang et al., 2006).

41 AaRSs and EF-1α perform two consecutive functions in translation:

aminoacylation of tRNA and subsequent delivery to the ribosome (Figure 1.11).

Although the molecular mechanism remains largely unknown, complex formation

between the elongation factor and an aaRS supports the direct hand-off of aa-tRNAs

synthesized by the aaRS directly to the ribosome via EF-1α without diffusion to the

cytoplasm. The link between aaRSs and EF-1α lends support to the notion of substrate

channeling to the ribosome. Further extending this role, EF-1α·GDP may then

complete the cycle by accepting uncharged tRNA from the ribosomal E site for another

round of aminoacylation by the associated aaRS (Petrushenko et al., 1997). This is evidenced by the unusual complex PheRS forms with EF-1α·GDP and uncharged

tRNA (Dibbelt & Zachau, 1981; Petrushenko et al., 1997; Petrushenko et al., 2002).

Beyond translation, EF-1α has also been implicated in chaperone-like functions,

protecting from denaturation and promoting renaturation of PheRS and SerRS (Caldas

et al., 1998; Gonen et al., 1994; Hotokezaka et al., 2002; Kudlicki et al., 1997; Lukash

et al., 2004). Through a concerted effort, EF-1α may associate with aaRSs in

eukaryotes to facilitate the delivery of aa-tRNA to the ribosome and to promote protein

stability.

42

EF-1α GTP

Figure 1.11. The two consecutive roles aaRSs and EF-1α play in translation. AaRSs and EF-1α perform two consecutive functions: aminoacylation of tRNA by the aaRS and subsequent delivery to the ribosomal A site by EF-1α. Adapted from Ataide & Ibba, 2006.

43 1.9. Multi-aminoacyl-tRNA complexes in archaea

A handful of binary complexes containing one aaRS and a second protein factor have been discovered in bacteria as described above (see section 1.4), although bacterial aaRSs are generally thought to function as stand-alone proteins. On the contrary, eukaryotes are known to harbor higher order complexes composed of multiple aaRS activities in association with a variety of cellular factors as described above, exemplifying the complex network of protein-protein interactions in higher organisms.

Less is known about the multimeric state of aaRSs in archaea, however, as only two reports of archaeal multi-aaRS complexes have been described and are presented below.

In an early report, several if not all aaRSs were observed to associate into a large multi-aminoacyl-tRNA synthetase complex (MSC) in the archaeon Haloarcula marismortui, providing precedence for the existence of higher order complexes containing aaRSs in archaea (Goldgur & Safro, 1994). More recently, ProRS has been found to associate in Methanocaldococcus jannaschii with a metabolic protein

(Mj1338) predicted to be related to the N5, N10-methylene tetrahydromethanopterin dehydrogenase family of enzymes that is involved in one-carbon metabolism in archaea

(Lipman et al., 2003). Although the function of Mj1338 has not been completely

resolved, the metabolic protein has been demonstrated to possess a general affinity for

tRNA (Lipman et al., 2003). The archaeal ProRS bound Mj1338 with a KD of about

1.4 µM, indicating stable protein-protein interactions. Although the formation of a higher order complex has not been verified, LysRS and AspRS each interacted

44 independently with Mj1338, suggesting a possible association with the archaeal

ProRS:Mj1338 complex.

As is the case for the higher order complex of aaRSs discovered in

H. marismortui, the biological function of complex formation between archaeal ProRS

and Mj1338 remains largely unknown, as complex formation did not stimulate

aminoacylation activity (Lipman et al., 2003). Although further experimentation would

be required to investigate the formation of a higher order complex in M. jannaschii,

these specific aaRSs (ProRS, LysRS, and AspRS) are of interest as their eukaryotic

counterparts are known to associate into a larger MSC in mammalian cells composed of

nine aaRS activities and three auxiliary proteins (see section 1.6; (Kerjan et al., 1994;

Mirande et al., 1985; Quevillon et al., 1999; Robinson et al., 2000). Further studies of archaeal complexes harboring aaRSs are now necessary to investigate the biological roles of these complexes in translation, which is the focus of the work presented below.

45

CHAPTER 2

FUNCTIONAL ASSOCIATION OF THREE AMINOACYL-tRNA

SYNTHETASES IN TRANSLATION

2.1. Introduction

The fidelity with which mRNA is decoded into protein is essential for maintenance of the genetic code and is dependent on aaRSs, the family of enzymes responsible for the specific coupling of amino acid and tRNA molecules. Once synthesized, the aminoacyl-tRNA (aa-tRNA) molecules are selectively bound and delivered to the ribosome by EF-1α, providing the growing polypeptide chain with substrates for protein synthesis. An intricate network of protein-protein interactions is required for efficient translation in archaea and eukaryotes (Lipman et al., 2003;

Mirande et al., 1985). In archaea, for example, preliminary evidence of multi-protein complexes harboring aaRSs has been revealed; in an early report, a large macromolecular complex harboring most if not all aaRSs in H. marismortui was observed (Goldgur & Safro, 1994). More recently, a second complex was discovered in M. janaschii, composed of ProRS and a metabolic protein (MJ1338; also possibly associating with LysRS and AspRS), although the biological role remains undefined as no change in aminoacylation was observed (Lipman et al., 2003).

46 Mammalian cells harbor a macromolecular complex composed of nine aaRS

activities (LysRS, LeuRS, GluProRS, IleRS, MetRS, GlnRS, ArgRS, and AspRS;

(Mirande et al., 1985; Norcum, 1991). Three auxiliary proteins complete the

mammalian MSC, playing roles in protein stability and tRNA binding (Ahn et al.,

2003; Kim et al., 2002; Quevillon & Mirande, 1996; Quevillon et al., 1997). Although

the protein components of the mammalian MSC were deciphered nearly 30 years ago,

the biological role of complex formation in translation remains largely undefined (Han

et al., 2006a; Kerjan et al., 1994; Mirande et al., 1985; Negrutskii & Deutscher, 1991;

Norcum & Warrington, 1998; Park et al., 2005b; Robinson et al., 2000). To shed some

light on the functional consequences of these associations, aaRS complexes were

investigated in archaea, since preliminary evidence of relatively simple archaeal multi-

enzyme complexes harboring aaRSs have been established. To investigate aaRS

complexes outside of the mammalian model system, a systematic search for archaeal

proteins interacting with Methanothermobacter thermautotrophicus LysRS and ProRS

was undertaken, the eukaryotic counterparts of which are known to associate in multi-enzyme complexes in mammalian cells.

2.2. Materials and methods

2.2.1. LysRS protein purification

A vector producing an N-terminally tagged His6 fusion derivative of

M. thermautotrophicus LysRS (MTH 1542; lysK) was prepared by inserting the

relevant PCR-amplified gene into the Escherichia coli expression plasmid pET15b

(Novagen). For the His6-LysRS construct, the forward primer

47 5’-CATATGAAGTTCACACACTTT-3’ and the reverse primer

5’-GGTGGTCTGCAGTCAGGCCTCCAGTCT-3’ were used, introducing NdeI and

PstI restriction digestion sites. Cloning into pET15b was done by isolating the

respective NdeI and PstI fragment of LysRS and ligating it into Nde1- and Pst1-

digested pET15b. His6-LysRS was produced by transforming E. coli BL21-RIL

(Stratagene) with pET15b-MtlysK. The resulting transformants were grown and induced to produce protein using the Over-night Express Auto-induction System 1

(Novagen) following the manufacturer’s protocol.

Cell free extract was produced by sonication of the cells in buffer A (50 mM

Hepes, pH 7.2, 25 mM KCl, 10 mM MgCl2, 5 mM dithiothreitol (DTT), and 10 %

glycerol) containing a protease inhibitor mixture tablet (Complete Mini, EDTA-free;

Roche Applied Science) followed by centrifugation at 75,000 xg for 20 min. To reduce

the amount of contaminating E. coli proteins, the supernatant was incubated at 55 ºC

for 10 min followed by a brief centrifugation to remove precipitated proteins and then

ultracentrifugation at 100,000 xg for 1 h. The supernatant from ultracentrifugation was

loaded onto a HiPrep 16/10 Q Sepharose FF column (GE Healthcare) equilibrated in

buffer A and extensively washed in the same buffer. The His6-LysRS was eluted with a

NaCl gradient (0 - 1 M) in the same buffer. All steps were performed at 4 ºC unless otherwise specified. Fractions containing His6-LysRS, as determined by aminoacylation activity, were pooled and concentrated by ultrafiltration (Amicon 30;

Millipore). The concentrated sample was further purified using gel filtration on a

Superose 12 column (GE Healthcare) equilibrated in buffer A containing 100 mM

NaCl. Fractions containing His6-LysRS, as determined by SDS-polyacrylamide gel

48 electrophoresis and Coomassie Brilliant Blue staining were pooled, concentrated by

ultrafiltration, and stored at -80 ºC.

2.2.2. ProRS and LeuRS protein purification

N-terminally tagged His6 fusion derivatives of M. thermautotrophicus ProRS

(MTH 611) and LeuRS (MTH 1508) were made by PCR amplification of the relevant genes. Templates were either genomic DNA or plasmids containing the relevant genes.

The PCR products were cloned into PCR-Blunt II-TOPO vector (Invitrogen) and sequenced prior to cloning into the E. coli expression plasmid pET11a (Novagen). For the His6-ProRS construct, forward and reverse primers

5’-CATATGCATCACCATCACCATCACCAGAACCTATCAAA-3’ and

5’TGATCATTAGCTAATATGTTC-3’ were used, respectively. Forward primer

5’-CATATGCATCACCATCACCATCACGATATTGAAAGAAAATGG-3’ and reverse primer 5’-TGATCATTATTCAAGGTATATGGCTGGCT-3’ were used for

His6-LeuRS. The oligonucleotide primers introduced NdeI and BclI restriction digest sites. Cloning into pET11a was performed by isolating the respective NdeI-BclI fragments and ligating them into NdeI-BamHI-digested pET11a vector DNA. Protein purification and production of ProRS and LeuRS was performed by transforming

BL21(DE3)RP or RIL strains (Stratagene) with pET11a containing the relevant inserts and growing the resulting strains using the Over-night Express Auto-induction System

1 (Novagen) according to the manufacturer’s instructions.

For His6-LeuRS, cell-free extract was prepared by sonication of E. coli cells in

lysis buffer (50 mM NaH2PO4, 300 mM NaCl) containing protease inhibitor mixture

49 tablet (Complete Mini, EDTA-free; Roche Applied Science) followed by centrifugation

at 75,000 xg for 20 min. To minimize contaminating E. coli proteins, a flocculation

step at 55 ºC for 10 min was performed prior to ultracentrifugation at 40,000 rpm for

1 h. The supernatant was then applied to a Ni2+-nitrilotriacetic acid Superflow column

(Qiagen) equilibrated in lysis buffer and extensively washed in the same buffer containing 10 mM imidazole. His6-LeuRS was eluted in the same buffer containing

250 mM imidazole. Fractions containing His6-LeuRS, judged by Coomassie Brilliant

Blue staining following SDS-PAGE separation, were pooled and buffer was exchanged

to buffer A using a HiPrep 26/10 desalting column (GE Healthcare). Samples were

concentrated and further purified to >95 % purity by ultrafiltration (Amicon Ultra-15;

30-kDa cut-off) as judged by Coomassie Brilliant Blue staining. Aliquots were stored

at -80 ºC.

Protein purification of His6-ProRS was performed as for His6-LeuRS, except

that the flocculation step was omitted. Fractions eluted and pooled from the

Ni2+-nitrilotriacetic acid Superflow column were diluted five-fold in water and subjected to ultrafiltration as above. Samples were then diluted five-fold in buffer A and applied to a Resource Q column (GE Healthcare) and eluted with a NaCl gradient

(0-500 mM) in the same buffer. Fractions containing ProRS, as monitored by

Coomassie Brilliant Blue staining following SDS-PAGE separation, were pooled,

subjected to ultrafiltration, aliquoted, and stored at -80 ºC. The concentrations of

LeuRS and ProRS were determined by active site titration as previously described

(Fersht et al., 1975).

50 2.2.3. Methanothermobacter thermautotrophicus total tRNA purification

To prepare total tRNA, M. thermautotrophicus cells (5 g; a gift from J. Reeve,

The Ohio State University) were resuspended in extraction buffer (20 mM Tris, pH 7.5,

20 mM magnesium acetate, 5 mM 2-mercaptoethanol, and a protease inhibitor mixture

tablet (Complete Mini, EDTA-free; Roche Applied Science) and extracted with acid-

buffered phenol (pH 4.5) as previously described (Polycarpo et al., 2003). DNA was partially removed by precipitation with 20 % (v/v) 2-propanol and centrifugation for

15 min at 4500 × g. The supernatant was adjusted to 60 % (v/v) 2-propanol and the total nucleic acids were harvested by centrifugation (20 min at 4500 × g). The pellet was suspended in water, chloroform extracted, and the unfractionated tRNA was recovered by ethanol precipitation. After centrifugation, the pellet was washed, dried briefly, and resuspended in 0.1 M MOPS (pH 7.0). tRNA was further purified on a

Qiagen-tip 10000 (Qiagen, Valencia, CA) column previously equilibrated with 80 ml

0.05 M MOPS (pH 7.0), 15 % isopropanol, 1 % Triton X-100. The unfractionated tRNA was applied to the tip, and the resin was washed with 0.05 M MOPS (pH 7.0),

0.2 M NaCl. The pure unfractionated tRNA was eluted with 0.05 M MOPS (pH 7.0),

0.75 M NaCl, 15 % ethanol, and A260 monitored. After elution, the fractions containing

the tRNA were precipitated with 1 volume of isopropanol at −20 °C overnight and

recovered by centrifugation. The total tRNA concentration was estimated

spectrophotometrically (A260/A280), and individual tRNA acceptor levels were

measured in plateau charging reactions (tRNALys = 0.41 pmol/µM;

tRNALeu = 0.24 pmol/µg; tRNAPro = 0.40 pmol/µg).

51 2.2.4. Co-purification of native LysRS, LeuRS, and ProRS via size exclusion and

anion exchange chromatography

M. thermautotrophicus cells (5g; a gift from J. Reeve, The Ohio State

University) were resuspended in buffer A (50 mM Hepes, pH 7.2, 25 mM KCl, 10 mM

MgCl2, 5 mM DTT, and 10 % glycerol) containing a protease inhibitor mixture tablet

(Complete Mini, EDTA-free; Roche Applied Science), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM N-α-(p-toluene sulfonyl)-L-arginine methyl ester to a final volume of 10 ml. The cells were then passaged twice through a French pressure cell, sonicated, and centrifuged at 48,000 xg for 30 min. The supernatant was then removed and centrifuged for 40 min at 100,000 xg. The resulting archaeal cell-free (S100) extract was then loaded onto a Sephacryl S300 26/60 column (GE Healthcare), previously equilibrated in buffer B (buffer A containing 150 mM KCl) and developed in the same buffer. All of the steps were performed at 4 ºC. The activities of the three aaRSs were assayed via aminoacylation in the presence of total M. thermautotrophicus tRNA and the corresponding radiolabeled amino acids (see section 2.2.9).

The fractions harboring the co-purified aaRS activities were pooled, concentrated by ultrafiltration (Amicon 30, Millipore), and applied to a HiPrep 16/10 Q

Sepharose FF column (GE Healthcare) anion exchange column, which was extensively washed and equilibrated in buffer B. All of the steps were performed at 4 ºC. The proteins were eluted with a KCl gradient (0.125 - 1.125 M) in the same buffer and the activities of the three aaRSs were assayed from each eluted fraction via aminoacylation in the presence of total M. thermautotrophicus tRNA and the corresponding radiolabeled amino acids.

52 2.2.5. Co-purification of His-tagged LysRS, LeuRS, and ProRS by size exclusion

chromatography

Heterologously expressed His-tagged LysRS, LeuRS, and ProRS proteins were

applied to a Superose 6 10/300 GL column (Amersham Biosciences) high performance

size exclusion column, which was extensively washed and equilibrated in buffer B. All

of the steps were performed at 4 ºC. The proteins were eluted in the same buffer and

the activities of the three aaRSs were assayed from each eluted fraction via

aminoacylation in the presence of total M. thermautotrophicus tRNA and the

corresponding radiolabeled amino acids (see section 2.2.9).

2.2.6. Fluorescent labeling of LeuRS

LeuRS was labeled with Alexa Fluor (AF) 488 tetrafluorophenyl ester

(Molecular Probes, Eugene, OR), which reacts with solvent exposed amines. LeuRS

was chosen for fluorescent labeling due to the significantly higher stability it displayed

over LysRS and ProRS for the labeling procedure. A solution of AF-488 was prepared

in DMSO, and the concentration was determined using the extinction coefficient

-1 -1 ε494 = 71,000 cm M according to the manufacturer’s protocol. Prior to labeling, the

LeuRS stock was applied to a 1 ml Sephadex G25 spin column (Amersham

Biosciences) equilibrated in a buffer containing 40 mM Hepes pH 7.5, 50 mM NaCl, and 50 mM KCl. LeuRS was labeled with AF-488 at a molar ratio of

1:25 LeuRS:fluorophore for 45 min at room temperature in the same buffer. Excess unreacted dye was immediately removed by passage through a 1 ml Sephadex G25 spin column pre-equilibrated in the same buffer. The labeled protein, LeuRS-AF, was

53 dialyzed in buffer A and then applied to a Microcon YM-50 concentrator (Amicon) to remove residual free dye. The final labeling stoichiometry was determined to be approximately 1:0.8 enzyme:fluorophore by Equation 1,

R = [A494 x (dilution factor)]/[ ε494 x Y) (Eq.1)

Where R is the molar ratio of dye to LeuRS, A494 represents the absorbance of labeled

protein solution at 494 nm, ε494 is the extinction coefficient of the dye, and Y is the final concentration of LeuRS-AF determined by active site titration (Fersht et al., 1975).

LeuRS-AF was visualized on a 10 % SDS-polyacrylamide gel and subjected to ultraviolet illumination, which confirmed that the final labeled product contained little or no free fluorophore. Prior to use in fluorescence anisotropy measurements, the activity of LeuRS-AF was verified by aminoacylation assays and protein concentrations determined via active site titration (data not shown; (Fersht et al., 1975)]).

2.2.7. Fluorescence anisotropy experiments: LeuRS with ProRS and LysRS

Equilibrium dissociation constants were determined by measuring the fluorescence anisotropy of LeuRS-AF as a function of increasing concentrations of an unlabeled protein. LeuRS-AF (100 nM) was incubated with increasing amounts of unlabeled protein for 20 min at room temperature in a buffer containing 50 mM Hepes pH 7.5, 250 mM KCl, 10 mM MgCl2, 125 mM glutaric acid, and 5 mM DTT. The

following concentration ranges of unlabeled protein were used: 25-2000 nM LysRS

and 25-5500 nM ProRS. Fluorescence anisotropy measurements were performed using 54 a spectrofluorometer (Fluorolog® Horiba Jobin Yvon). The excitation and emission wavelengths were 490 and 519, respectively, with slit widths of 5 nm. Anisotropy was measured using the time-based function for 30 s (integration time = 1 s; resolution = 8 s) and the data were averaged. All measurements were carried out at least three times. The titration curves were fit to Equation 2, which assumes a 1:1 binding stoichiometry as previously described (An & Musier-Forsyth, 2005),

2 1/2 A = Amin + [(Y+S+KD) - {(Y+S+KD) - (4YS)} ] · (Amax - Amin)/(2Y) (Eq. 2)

Where A is the measured anisotropy at a particular total concentration of unlabeled

protein, S, and LeuRS-AF (Y), Amin is the minimum anisotropy, Amax is the maximum

anisotropy, and KD is the dissociation constant.

2.2.8. Preparation of in vitro transcribed tRNA

In vitro T7 RNA polymerase run-off transcription of M. thermautotrophicus

tRNAPro, tRNALys, and tRNALeu was prepared as described (Mursinna et al., 2001).

The tRNA transcript was purified on a denaturing 12 % polyacrylamide gel and recovered by UV-shadowing and subsequent elution from the polyacrylamide gel piece by electrophoresis. The tRNA was then phenol and chloroform extracted, ethanol precipitated, and resuspended in DEPC-treated H2O containing 2 mM MgCl2. The

transcribed tRNA was re-folded by incubation for 1 min at 80 °C, followed by slow

cooling to 25 °C. In vitro transcribed M. thermautotrophicus tRNALys was inactive in aminoacylation (data not shown).

55 2.2.9. Aminoacylation assays for archaeal LysRS, LeuRS, and ProRS

Aminoacylation assays were performed at 50 ºC. A pre-reaction mixture

containing 100 mM Hepes, pH 7.5, 250 mM KCl, 10 mM MgCl2, 10 mM DTT,

50 µg/ml bovine serum albumin, 6 mg/ml M. thermautotrophicus total tRNA, or in vitro transcribed tRNA at concentrations indicated, and aaRSs at concentrations indicated for specific experiments were pre-incubated for 20 min at room temperature.

The appropriate radiolabeled amino acid was then added to the mixture and the temperature increased to 50 ºC. After 1 min, 5 mM ATP was added to start the reaction. Aliquots were removed at times indicated for specific experiments, spotted onto 3MM paper presoaked in 5 % trichloracetic acid (TCA; w/v), washed in TCA, and

14 radioactivity counted. For lysine KM determination, L-[ C]lysine (312 mCi/mmol;

Perkin Elmer Life Sciences) was added at concentrations varying between 0.2 and

Lys 3 times KM. For tRNA KM determination, total tRNA was added at a final

Lys concentration of tRNA varying between 0.2- and 3-times the KM. Due to the relatively low activity of in vitro transcribed tRNALeu, saturation conditions could not

14 be achieved, and kcat/KM was estimated directly. L-[U- C]leucine (331 mCi/mmol;

3 Perkin Elmer Life Sciences). For ProRS KM determination, L-[ H]proline

(276.5 mCi/mmol; Perkin Elmer Life Sciences) was added at concentrations varying

Pro between 0.2- and 5-times KM. For tRNA KM determination, tRNA was added at

concentrations varying between 0.2- and 5-times the KM.

56 2.3. Results

2.3.1. Yeast two-hybrid analysis of proteins interacting with LysRS and ProRS

Two yeast two-hybrid screens of an archaeal M. thermautotrophicus cDNA library were previously performed, screening for proteins interacting with LysRS and ProRS as bait proteins based on continuous growth on selective media as previously described

(Table 2.1; see section 3.2.2; [(Praetorius-Ibba et al., 2005; Praetorius-Ibba et al., 2007;

Kasiviswanathan et al., 2006)]). Proteins identified as potentially interacting with

LysRS and ProRS may be divided into four main categories based on functional annotation: methanogenesis, protein binding and modification, translation, and those of unknown function (Tables 2.2 and 2.3). The only known component of protein synthesis identified in both screens as a potentially-positive interacting protein was

LeuRS. Sequencing of the LeuRS-encoded clones from the two screens identified an

N-terminal fragment of LeuRS as the interacting partner of LysRS, while a C-terminal fragment of LeuRS was identified as interacting with ProRS. This complementary finding suggested how the three aaRSs could form a ternary complex mediated by

LeuRS (see Chapter 4).

57

r rd e o h t in a v d a d l l r d cto r n r o a cti ow a r d st d d le ntr sta ve 3 n d. -, n t o an t 0 n a or or G contro dar c te t o s n st ct c a

or n a e e c bai t n cti t n o i on se c i a ctor o v v t i r s e e en). c tio e e MaV2 ct t ey ey v a g the v on s r r p r a n e r e y ey p p cti ey e t a tro e i t n n a i t i i i o int n d in s pr

pr a d as ind v n escrip n ty k i te de RS RS evi p D ong n o ea oder o inter e m ; I Leu E M Leu N Str W Empty m cer

th phe . em

enc

t

S

s

ly ow

d y

r e

upple A G s

rate rm d s a + + + + o - p /- + + + + + + +/ + + + + + + se 5-FO nsf ( sRS. ophan % y S L .2 -tra 0 o wo-hybri sR t trypt y nd a + L nd r a S

o th. Quest S

oR ine ro ow

Pr oR r r Growth of c (P T g h

P

A t

i - g leuc D 3 n - w i een k

A M ctor). S

+ w

+

- t strong /- + / + + + + -- e m lac ve + + + + ns uR

+ i 5 y e

+ b 2

s n

; + pre

dium : L io s, + th i

l prote

H me the act - ow o ions r e

n r t

t e i t c

g le d n

a + - i p te - r + /

- - / a + - - contr

+ + r r

m + + ra te

ode o

o U

n n

- de

c

a f o

ee i

id i tic

m tw

br +

RS y

nthe RS u

e med e

L th; + ns be iv sy -

o n

y inserts (enc T w 6 ct i 6 t e S o l 8 86-Leu c E e C C º ra PC8

ast two-h PC P s /p /pD e p p 0 n / ak gro S S 3 d librar e S

o t n Y R RS/ a w s s es s 1. y h

for inte L -ProR -ProR id ; + yp - -Ly 8 u u l B l C l D l A eu) a t

h u u e e 4 t o o o e 2. e e ro l L L t r BL dia bl ntr ntr ntr n eno asm e a h Pl Co Co Co Co pDB pDB pDBl pDB T m p pD afte grow

58 Function ORF Description

Methanogenesis: * MTH1159 N5-methyl-tetrahydromethanopterin: CoM methyltransferase, a-sub * MTH1160 N5-methyl-tetrahydromethanopterin: CoM methyltransferase, b-sub * MTH1163 N5-methyl-tetrahydromethanopterin: CoM methyltransferase, e-sub MTH1300 Co-F420-reducing hydrogenase, a-sub MTH1878 CoB-CoM heterodisulfide reductase Fe-S,C-sub

Protein binding and modification: * MTH357 Transglutaminase * MTH412 Transglutaminase MTH785 La protease MTH1623 Oligosaccharyl transferase STT3

Translation: MTH1508 Leucyl-tRNA synthetase

Other: MTH736 Carbomoyl phosphate synthase, large sub MTH802 Aspartokinase II -sub MTH957 ATP synthase subunit C * MTH674 Unknown function

Table 2.2. Proteins identified as interacting with ProRS. *Identified in previous screens (Kasiviswanathan et al., 2006).

Function ORF Description Methanogenesis: * MTH1130 Methyl-CoM RDase II, γ-sub. * MTH1134 Methyl viologen-reducing hydrogenase * MTH1161 N5-methyl-tetrahydromethanopterin: CoM methyltransferase, c-sub * MTH1165 Methyl CoM RDase I, γ-sub MTH1166 Methyl CoM RDase c-sub * MTH1168 Methyl CoM RDase I, β-sub MTH1300 Co-F420 Reducing hydrogenase MTH1752 F420-dependent N5,N10-methylene tetrahydromethanopterin reductase MTH1878 CoB--CoM heterodisulfide reductase Fe-S,C-sub

Protein binding and modification: MTH32 Centromere/microtubule binding protein * MTH357 Transglutaminase * MTH412 Transglutaminase MTH678 Prefoldin beta subunit β-sub MTH794 Chaperonin (thermosome β-sub)

Translation: MTH1508 Leucyl-tRNA synthetase

Other: MTH810 Putative ski2-type helicase MTH1588 Ferripyochelin binding protein MTH1913 Putative membrane protein

Unknown function: MTH247, *MTH479, MTH545, MTH609, *MTH674, MTH1191

Table 2.3. Proteins identified as interacting with LysRS. *Identified in previous screens (Kasiviswanathan et al., 2006).

59 2.3.2. Association of LysRS, LeuRS, and ProRS in vivo and in vitro

To investigate the possible interactions between archaeal LysRS, LeuRS, and

ProRS in vivo, co-purification of the corresponding activities from cell-free extracts was monitored. An S100 fraction was prepared from M. thermautotrophicus and applied to an S300 26/60 gel filtration column (GE Healthcare). The activities of native LysRS, LeuRS, and ProRS were monitored in each eluted fraction via aminoacylation in the presence of the corresponding radiolabeled amino acid and purified M. thermautotrophicus total tRNA. The aminoacylation activities of LysRS,

LeuRS, and ProRS co-eluted from the gel filtration column (Figure 2.1 A), suggesting specific complex formation in vivo. Calibration of the column with molecular mass standards indicated that the peak in the three aminoacylation activities corresponds to a molecular mass of roughly 200 - 255 kDa, which is broadly consistent with a 1:1:1 stoichiometry based on the predicted molecular masses of each aaRS (monomeric

LysRS ~64 kDa; monomeric LeuRS ~108 kDa; and dimeric ProRS ~112 kDa; predicted total molecular mass ~280 kDa).

In an effort to further examine the formation of an archaeal aaRS complex in vivo, fractions containing the co-eluted aaRSs (eluting between 145 and 200 ml) from the S300 26/60 gel filtration column were pooled and applied to a HiPrep 16/10 Q

Sepharose FF anion exchange column (GE Healthcare), which was extensively washed and equilibrated. The proteins were eluted with a KCl gradient (0.125 - 1.125 M) and the activities of the native LysRS, LeuRS, and ProRS enzymes were assayed via aminoacylation in the presence of total purified M. thermautotrophicus tRNA and the corresponding radiolabeled amino acid. LysRS, LeuRS, and ProRS again eluted

60 together, this time showing co-activity peaks both early and late in the applied KCl

gradient, suggesting partial dissociation of the complex under these conditions (Figure

2.1 B). Attempts to further co-purify the LysRS:LeuRS:ProRS complex by applying

the active pools to a cation exchange column were not successful (data not shown).

Similar difficulties have also been reported for the co-purification of the larger multi-

aaRS complex in mammalian cells (Mirande et al., 1982a; Quevillon et al., 1999).

Co-purification of the putative LysRS:LeuRS:ProRS complex was investigated

in vitro using the corresponding heterologously expressed and purified His-tagged proteins. Following co-incubation of the three His-tagged aaRSs at room temperature, the possible formation of a ternary complex was investigated by gel filtration chromatography (Superose 12 gel filtration column; GE Healthcare). Eluted fractions were then subjected to SDS-PAGE analysis and the presence of each protein component visualized by Coomassie Brilliant Blue staining. A peak was observed with an approximate molecular mass of 600 kDa, which is broadly consistent with a complex stoichiometry of 2:2:2 (predicted total molecular mass ~560 kDa) and in agreement with the approximately equal amounts of each protein observed in the corresponding fraction based on visualization by SDS-PAGE analysis (Figure 2.2). In separate experiments, no evidence for the reconstitution of a possible interaction between LysRS and ProRS was observed, consistent with yeast two-hybrid data

(M. Ibba; data not shown).

61

1.4 1.2

1.2 l) 1

Lys o l) l) mol) o o m m 1 Leu m p p ( (

(p 0.8

A Pro

N 0.8 R RNA t t l- tRNA (p

l- 0.6 y y l- c c

0.6 l-tRNA a a o cy o n cy i in 0.4 m

0.4 m A A inoa 0.2 0.2 Aminoa Am 0 0 50 100 150 200 250 300 350 400 1 Elution volume (ml) Elution volume (ml)

1.2 1.2 Lys

l) 1 Leu 1 o l)

o Pro m m p (

0.8 0.8 ) ) A M ( N M l ( NA (p R C t l- R 0.6 0.6 K y c l-t a KCl o cy in 0.4 0.4 m A inoa

m 0.2 0.2 A

0 0 0 10 20 30 40 50 60 70 80 Elution volume (ml) Elution volume (ml)

Figure 2.1. Co-purification of LysRS, LeuRS, and ProRS. A. M. thermautotrophicus cell-free extracts were applied to a Sephacryl S300 column, and the aminoacylation activities were monitored in the eluted fractions. B. Active fractions from A were pooled and applied to a Q-Sepharose column and extensively washed prior to development with a KCl gradient (see text for detail). Aminoacylation activities were monitored in the eluted fractions. , Lys-tRNA; U Leu-tRNA; Pro-tRNA▲.

62

MW standards Proteins of interest

Figure 2.2. Gel filtration chromatography of His-tagged LysRS, LeuRS, and ProRS. LysRS, LeuRS, and ProRS were applied either alone or in combination after preincubation to a Superose 12 column. Calibration curves show the elution positions of both individual and combined proteins. ●, molecular mass standards; ▲, aaRS samples. SDS-PAGE (10 %) analysis of the ~600 kDa fraction is shown in the inset.

63

2.3.3. Determination of dissociation constants within the archaeal multi-

aminoacyl-tRNA synthetase complex

To investigate the pair-wise binding affinities of the aaRSs associated in the

archaeal complex, fluorescence anisotropy experiments were performed, measuring the

KD values for LeuRS binding to LysRS and ProRS. Briefly, LeuRS was labeled with an amine-reactive extrinsic fluorophore AF-488 (Molecular Probes, Eugene, OR).

LeuRS was chosen for fluorescent labeling due to the significantly higher stability over

LysRS and ProRS for the labeling procedure. Fluorescence anisotropy experiments

were performed in the presence of fluorescently labeled LeuRS (LeuRS-AF) and

increasing concentrations of unlabeled partner protein (LysRS or ProRS). Protein-

protein interactions result in a significant increase in anisotropy from which

dissociation constants are calculated. Prior to use in experiments, the activity of

fluorescently labeled LeuRS-AF was verified by aminoacylation assays and protein

concentrations determined by active site titration (Fersht et al., 1975).

In the presence of LeuRS-AF and increasing concentrations of unlabeled

ProRS, a significant increase in anisotropy was observed. Data were fit to a binding

curve assuming a 1:1 stoichiometry and a KD of 970 ± 10 nM was calculated (Figure

2.3 A). Similarly, titration of LeuRS-AF with LysRS resulted in a binding constant of

270 ± 50 nM (Figure 2.3 B). Although it would be of interest to measure the binding

affinity in the presence of tRNA, the use of in vitro transcribed tRNALys was not a viable option because the tRNA is inactive in aminoacylation. These results are in accordance with the biophysical studies described above, supporting formation of a

64 stable multi-aaRS complex in M. thermautotrophicus composed of LysRS, LeuRS, and

ProRS.

LeuRS-AF + ProRS LeuRS-AF + LysRS

KD = 970 ± 10 nM KD = 270 ± 50 nM

Figure 2.3. Fluorescence anisotropy experiments: LeuRS with ProRS and LysRS. The binding of Alexa Fluor-labeled LeuRS to ProRS (A) and LysRS (B) was measured using 100 nM of LeuRS-AF as a function of increasing concentrations of unlabeled protein. A representative data set is shown, with values representing the means and corresponding standard deviations from three independent experiments.

2.3.4. Effects of association of LysRS, LeuRS, and ProRS on aminoacylation

The potential impact of complex formation was investigated by determining the steady-state aminoacylation parameters of each aaRS in the presence or absence of the other proteins associated in the MSC. In the presence of LeuRS, the catalytic efficiencies of aminoacylation by ProRS and LysRS were enhanced five-fold and three- fold, respectively, while no significant changes in the kinetics of aminoacylation by

LeuRS were observed (Tables 2.4 and 2.5, respectively). No further changes were identified upon addition of the aaRSs in alternative combinations (Tables 2.5 and 2.6).

Steady-state aminoacylation kinetic parameters for LysRS in the presence or absence of

Lys LeuRS indicated that complex formation specifically decreased the KM for tRNA by 65 Lys three-fold, but had no effect on the KM for lysine or the synthesis rate of Lys-tRNA

(kcat). The addition of ProRS had a less pronounced effect on steady-state

Lys aminoacylation by LysRS, decreasing the KM for tRNA by about 30 %. The addition

of LeuRS or ProRS did not lead to any significant changes in the KM for lysine of

LysRS, suggesting that the interactions between the three aaRSs specifically improve tRNA binding. Taken together, this indicated the possible role of an archaeal MSC comprised of three aaRSs in which LeuRS improves the catalytic efficiencies of tRNA aminoacylation by both LysRS and ProRS.

1 2 Pro Enzyme Additions KM Pro Km tRNA kcat kcat/KM tRNA (µM) (µM) (min-1) (relative) ProRS None ND3 4.1 ± 0.9 1.8 ± 0.2 1 ProRS LeuRS ND 2.2 ± 0.6 5.1 ± 0.4 5 ProRS BSA 67 ± 10 2.4 ± 0.4 2.9 ± 0.1 3 ProRS LeuRS, BSA 61 ± 17 1.4 ± 0.1 6.9 ± 0.2 12

Table 2.4. Steady-state aminoacylation kinetics of M. thermautotrophicus ProRS. 1Enzymes were added at a final concentration of 40 nM. 2Other enzyme components at 400 nM each. 3ND, not determined.

1 2 Lys Enzyme Additions KM Lys Km tRNA kcat kcat/KM tRNA (µM) (µM) (min-1) (relative) LysRS None 6.6 ± 1.6 0.74 ± 0.15 0.86 ± 0.1 1 LysRS LeuRS 6.0 ± 2.0 0.24 ± 0.02 0.91 ± 0.03 3.3 LysRS ProRS 7.9 ± 0.9 0.51 ± 0.13 0.63 ± 0.08 1.1

Table 2.5. Steady-state aminoacylation kinetics of M. thermautotrophicus LysRS. 1Enzymes were added at a final concentration of 40 nM. 2Other enzyme components at 400 nM each.

66 1 2 Leu Enzyme Addition(s) kcat/KM Leu kcat/KM tRNA (sec-1 µM-1) (sec-1 µM-1) LeuRS BSA 0.35 ± 0.07 0.75 ± 0.08 LeuRS ProRS, BSA 0.37 ± 0.09 0.75 ± 0.03

Table 2.6. Steady-state aminoacylation kinetics of M. thermautotrophicus LeuRS. 1Enzymes were added at a final concentration of 40 nM. 2Other enzyme components at 400 nM each.

2.4. Discussion

2.4.1. Identification of LysRS- and ProRS-interacting proteins via yeast two- hybrid analysis

A MSC composed of LysRS, LeuRS, and ProRS was identified via yeast two- hybrid screening of an M. thermautotrophicus cDNA library with LysRS and ProRS as bait proteins (Table 2.1). Stable complex formation between LysRS, LeuRS, and

ProRS was confirmed in vitro and in vivo via co-purification experiments of the corresponding His-tagged proteins and from archaeal cell-free extracts, respectively

(Figures 2.1 and 2.2). Although the precise stoichiometry of the archaeal MSC remains debatable, yeast two-hybrid and biophysical data indicate the existence of an aaRS ternary complex in archaea with the composition LysRS•(N-)LeuRS(-C)•ProRS.

Lending support to the formation of a stable archaeal MSC, pair-wise binding affinities were determined from fluorescence anisotropy studies indicating that LeuRS bound

LysRS and ProRS with comparable KDs of about 0.3-0.9 µM (Figure 2.3), which is

five- to 10-fold lower than predicted cellular concentrations for the aaRS enzymes in

bacteria (Jakubowski & Goldman, 1984). If M. thermautotrophicus harbors similar

67 cellular concentrations of LysRS, LeuRS, and ProRS, the majority of these enzymes

would be expected to be found in the ternary complex based on the experimental KD

values. Further, these binding affinities are comparable to the bacterial ProRS:YbaK

complex (An & Musier-Forsyth, 2005), and are comparable to the known pair-wise

binding affinities between aaRSs and auxiliary proteins within the mammalian MSC

(Robinson et al., 2000).

Neither of the yeast two-hybrid screens revealed an interaction with H2-forming

N5-N10-methylene tetrahydromethanopterin dehydrogenase (Mj1338), a protein

previously shown to associate with ProRS in M. jannaschii (Lipman et al., 2003).

Although this protein involved in one-carbon metabolism in archaea was not found,

metabolically-related proteins were detected via yeast two-hybrid analysis with LysRS

and ProRS, providing further evidence of a possible association between protein

synthesis and metabolism in archaea as was originally proposed in M. jannaschii. The

significance of an interaction between an aaRS and the metabolic protein Mj1338

remains unclear; recent proteomics studies in other methanogens do not indicate a

regulatory role in methanogenesis, suggesting that the interaction may impact other

aspects of metabolism (Xia et al., 2006). Further experimentation would be required to

investigate a potential role of aaRSs in methanogenesis.

2.4.2. Functional consequences of aaRS complexes in translation

The functional consequences of complex formation were investigated by

determining the steady-state aminoacylation parameters of each aaRS alone or in the

presence of the other components of the archaeal MSC. In the presence of LeuRS, the

68 catalytic efficiencies of aminoacylation by LysRS and ProRS were enhanced three-fold and five-fold, respectively, while no significant changes in the kinetics of aminoacylation by LeuRS were observed (Tables 2.4 - 2.6). This indicates the possible role of an archaeal MSC comprised of three aaRSs in which LeuRS improves the catalytic efficiencies of tRNA aminoacylation by both LysRS and ProRS.

Interestingly, LysRS, LeuRS, and ProRS are all components of the larger MSC discovered in mammalian cells, although a few distinctions between the archaeal and eukaryotic counterparts exist (Figure 2.4). Although both the archaeal and eukaryotic

LysRSs function to covalently link lysine to tRNALys, the archaeal LysRS belongs to the class I family of aaRSs and exists as a monomer, whereas the eukaryotic LysRS is a class II dimer (Guo et al., 2005; Han et al., 2006a; Praetorius-Ibba & Ibba, 2003).

Another distinction between human and archaeal aaRSs is ProRS. While archaeal

ProRS exists as a singular protein, the catalytic domain of human ProRS is fused to

GluRS via a linker region, forming the bifunctional GluProRS characteristic of higher eukaryotes (Berthonneau & Mirande, 2000).

Mammalian MSC Archaeal MSC

GlnRS ↑ Lysylation 3-fold Ar gRS p43 MetRS p18 LysRS LeuRS AspRS LysRS LeuRS AspRS LysRS p38 ProRS GluProRS IleRS ↑ Prolylation 5-fold

Figure 2.4. Multi-aaRS complexes in H. sapiens and M. thermautotrophicus. 69 Several eukaryotic aaRSs harbor N- or C-terminal appended domains, which are

generally absent from their archaeal counterparts (Mirande, 1991). In several cases,

such as LeuRS, these appended domains are known to mediate protein-protein

interactions within the MSC. Human LeuRS and ProRS are believed to be anchored to

the auxiliary protein factor p38, which acts as a core scaffolding protein and is essential

for complex assembly and stability (Ahn et al., 2003; Kim et al., 2002; Quevillon et al.,

1999; Robinson et al., 2000; Wolfe et al., 2005). Another auxiliary factor of the

mammalian MSC, p43, has also been implicated as a general tRNA-binding protein and

core component of the complex (Simos et al., 1996; Wolfe et al., 2003). Although homologues of p43 have been identified in yeast (Arc1p) and bacteria (Trbp111), no archaeal orthologs of the MSC accessory proteins p38, p43, or p18 have been identified to date (Morales et al., 1999; Simos et al., 1996). Whether other unidentified proteins exist in archaea that harbor the same functions as the three mammalian accessory proteins is so far unknown.

If the accessory proteins associated with the mammalian MSC are absent in archaea, the functions of these factors may instead lie within the aaRSs themselves.

With this in mind, LeuRS may act as a scaffolding protein to stabilize the archaeal

MSC, enhancing the catalytic activities of LysRS and ProRS in M. thermautotrophicus.

Whereas the mechanisms underlying this effect remain unknown, our findings suggest that direct interactions between aaRSs, as seen extensively in higher eukaryotes, could potentially provide a novel means to directly improve aminoacylation efficiency.

Taken together, the presence of LysRS, LeuRS, and ProRS in both mammalian and archaeal complexes suggests that perhaps the smaller complex discovered in archaea is

70 an evolutionary precursor of the macromolecular MSC conserved in chordates. Further studies are now necessary to investigate the presence of additional protein factors in the

MSCs. In fact, the true identity of all protein components within the complex that occur in vivo may be masked, possibly due to the fragility of the complex, loosely associated peripheral proteins, or transient interactions that disassociate during co-purification procedures.

Although the stably associated components of the mammalian MSC are known, the precise function of placing the aaRS in this complex is less-defined. It has been suggested that, similar to the Arc1p:GluRS:MetRS complex discovered in S. cerevisiae, complex formation may increase protein stability or restriction of the associated aaRSs to the cytoplasm (Galani et al., 2001). Recently, it has been demonstrated in human cells that the aaRSs associated with the mammalian MSC may provide a sequestered pool of aa-tRNAs specifically for utilization in protein synthesis (Kyriacou &

Deutscher, 2008). Mammalian aaRSs have also been observed to co-migrate with polysomes, indicating a coordinated mechanism to ensure efficient translation

(Popenko et al., 1994). Taken together with the biological role of the smaller archaeal

MSC, the mammalian MSC may also function to enhance aminoacylation of the associated aaRSs.

71

CHAPTER 3

AN AMINOACYL-tRNA SYNTHETASE:ELONGATION FACTOR COMPLEX

FOR SUBSTRATE CHANNELING IN TRANSLATION

3.1. Introduction

Efficient translation relies on aaRSs to correctly link amino acid and tRNA molecules. Once synthesized, aa-tRNAs are selectively bound and ushered to the ribosome during translation elongation by translation factor EF-1α. Upon codon- anticodon recognition on the ribosome, GTP is hydrolyzed, releasing the GDP-bound form of EF-1α. The nucleotide exchange factor then functions to recycle GDP to GTP, permitting EF-1α to pursue another round of aa-tRNA selection.

In higher eukaryotes, aaRSs are known to associate with additional protein factors such as EF-1α, which provides a direct link between consecutive steps in translation: aminoacylation and subsequent delivery to the ribosome. Stable complex formation between human ValRS and the human elongation factor lend support to this notion

(Negrutskii & Deutscher, 1991; Stapulionis & Deutscher, 1995). Although the stoichiometry of the complex is debated, the presence of excess EF-1α and GTP enhanced the catalytic efficiency of human ValRS almost two-fold (Negrutskii et al.,

1999). This increase in the catalytic efficiency was not observed in the presence of

EF-1α·GDP or bacterial EF-Tu·GTP, indicating that the increased levels of

72 Val-tRNAVal were due to improved catalysis by ValRS. It is clear that protein-protein interactions within the translational machinery of archaea and eukaryotes are important to facilitate the process of protein synthesis, as was observed for the

LysRS:LeuRS:ProRS complex discovered in M. thermautotrophicus and the macromolecular MSC observed in higher eukaryotes. To investigate the role of archaeal EF-1α in substrate channeling, a yeast two-hybrid approach was undertaken to identify translational proteins associated with EF-1α in archaea.

3.2. Materials and methods

3.2.1. Yeast two-hybrid strains, plasmid construction, and cDNA library

The S. cerevisiae yeast host strain MaV203 (MATα leu2-3, 112, trp 1-901, his3∆200, ade2-101, gal4∆, gal80∆, SPAL10::URA3; GAL1::lacZ, HIS3UAS

GAL1::HIS3@LYS2, can1R, cyh2R), the bait vector pDBLeu, and the prey vector

pDEST22 were from the ProQuest two-hybrid system (Invitrogen). Media preparation

and transformation of MaV203 with the bait vector pDBLeu and prey vector pDEST22

were performed according to the manual for ProQuest Two-Hybrid System (Invitrogen)

and as described. To construct the yeast two-hybrid bait vector containing the full- length M. thermautotrophicus tuf gene (encoding EF-1α; MTH1058), the corresponding sequence was isolated by PCR using genomic M. thermautotrophicus

DNA as template, forward primer 5’-GTCGACCATGGCTAAAGA-3’ and reverse primer 5’-GCTAGCTTATTTTGCTGG-3’ flanked by SalI and NheI sites. The tuf PCR

product was cloned into PCR-Blunt II-TOPO vector (Invitrogen), sequenced, and

subsequently sub-cloned into the yeast ProQuest Two-Hybrid bait vector pDBLeu 73 using the SalI and NheI restriction sites. The resulting pDBLeu-tuf vector harbors the full-length gene encoding EF-1α fused in frame to the Gal4 DNA-binding domain

(DBD) encoded in the bait vector, which also contains a leucine biosynthesis cassette.

The M. thermautotrophicus cDNA library was generated by Christian Gruber and Mark Smith (Invitrogen) using random priming of total RNA from

M. thermautotrophicus. The resulting cDNA was then directionally cloned into the pDEST22 yeast two-hybrid prey vector, which fuses each cDNA to the Gal4 transcriptional activator domain (AD). pDEST22 also contains a tryptophan biosynthesis cassette. The resulting cDNA library contained 2.6 x 106 clones, which represents a >1000-fold coverage of the genome, with an average insert size of

0.4 - 2 kb.

3.2.2. Yeast two-hybrid screening

The bait vector pDBLeu-tuf (encoding EF-1α) was co-transformed with the

M. thermautotrophicus cDNA library into the S. cerevisiae host strain MaV203, which is auxotrophic for both leucine and tryptophan. Potential positive clones were selected by plating transformants on selective complete (SC) medium lacking tryptophan and leucine (SC -W-L) to screen for the presence of both vectors and determine the efficiency of transformation. Protein-protein interactions between the bait EF-1α and the prey encoded by the cDNA library were identified phenotypically, utilizing a

histidine biosynthesis reporter gene (his3). 3-aminotriazole (3-AT), a specific inhibitor of the his3 gene product, was titrated to determine the minimal inhibitory concentration to inhibit basal expression of the his3 gene, which is essential to reduce the number of 74 false positives in the screen. At 25 mM 3-AT, which was sufficient to reduce the basal

expression of his3, growth of Control A (negative control - two proteins with no

interaction) is inhibited, while Control B (weak interaction) and Control C (strong

interaction) are able to continuously grow on SC -Leucine -Tryptophan -Histidine

+25 mM 3-AT (Invitrogen ProQuest two-hybrid system). Potential positive clones

were selected by plating transformants on SC -Leucine -Tryptophan -Histidine +25 mM

3-AT and incubating plates at 30 ºC for 3-10 days, including replica cleaning as

described (ProQuest two-hybrid system). The transformation plates were then replica-

plated onto SC -Leucine -Tryptophan +25 mM 3-AT and incubated as above.

Transformants displaying consistent growth were streaked for isolation of single

colonies, which were then re-tested for growth on SC -Leucine -Tryptophan -Histidine

+10, 25, and 35 mM 3-AT. Isolated colonies showing consistent growth were also

tested for the protein-protein interaction phenotype utilizing the ura3 reporter gene,

whose gene product is essential for uracil biosynthesis and also catalyzes the

conversion of 5-fluoroorotic acid (5-FOA) into a toxic compound. Expression of ura3

due to protein-protein interactions between bait and prey results in reduced or no

growth of MaV203 on SC -Leucine -Tryptophan -Uracil +0.2 % 5-FOA medium

(Invitrogen ProQuest two-hybrid system), correlating to the strength of interaction.

A total of 1.4 x 105 transformants were screened for the protein-protein interaction phenotype and approximately 100 clones were selected for plasmid isolation and sequencing. Isolation of positive clones for sequencing was performed by growing the co-transformants in SC -Tryptophan medium followed by plating on SC -Tryptophan to isolate colonies harboring only the prey vector. Each of these colonies were then

75 grown in liquid SC -Tryptophan medium, prey vector extracted, and transformed into

E. coli for isolation and plasmid purification. Prey vector cDNA inserts were

sequenced using the oligonucleotide corresponding to the portion of the prey vector that

reads into the 5’-end of the insert.

3.2.3. Archaeal EF-1α, AlaRS, and bacterial EF-Tu protein production and

purification

His6 fusion derivatives of LeuRS, LysRS, and ProRS (MTH1508, MTH 1542, and MTH611, respectively) were prepared as previously described. C-terminal His6

tagged fusion derivatives of EF-1α and AlaRS (MTH1683) were prepared by inserting

the corresponding PCR amplified genes into pET11a and pET33b vectors, respectively.

For the His6-EF-1α construct, the forward primer

5’-CATATGGCTAAAGAAAAAGAACACATGA-3’ and the reverse primer

5’-TGCTCTTCCGCATTTTGCTGGTACGAGGTCTATG-3’ were used. Cloning

EF-1α into pET11a was done by isolating the respective NdeI and SapI fragment and

ligating into NdeI and SapI digested pET11a. For the His6-AlaRS construct, forward primer 5’-GCTAGCATGATTACCATGTCCCATCAGCTTGAA-3’ and reverse primer 5’-GCGGCCGCCCTTCCTCACAGTACTGAGTGCAGCT-3’ were used.

Cloning AlaRS into pET33b was done by isolating the respective NheI and NotI

fragment and ligating into NheI and NotI digested pET33b.

His6-LeuRS, His6-ProRS, and His6-LysRS were produced and purified as previously described. His6-AlaRS was produced by transforming E. coli BL21-RIL

(Stratagene) with pET33b-alaS. The resulting transformants were grown and induced 76 to produce protein using the Overnight Express Auto-induction System 1 (Novagen)

following the manufacturer’s protocol. Cell-free extract was produced by passing cells

in buffer A (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 10 mM imidazole)

containing a protease inhibitor mixture tablet (Complete Mini, EDTA-free; Roche

Applied Science) through a French pressure cell followed by centrifugation at

70,000 xg for 45 min.

To reduce the amount of contaminating E. coli proteins, the supernatant was

incubated at 60 °C for 10 min followed by ultracentrifugation at 100,000 xg for 1 hour.

2+ The supernatant from ultracentrifugation was loaded onto a Ni-NTA column, washed extensively with buffer A, and eluted with an imidazole gradient (0-250 mM) in the same buffer. His6-EF-1α was produced by transforming E. coli BL21-RIL with

pET11a-tuf and purified in a manner identical to AlaRS with the exception that

buffer A included the addition of 10 µM GDP to enhance protein stability. Fractions

containing His6-EF-1α and His6-AlaRS, as determined by SDS-PAGE Coomassie

Brilliant Blue staining were pooled, concentrated by ultrafiltration (Amicon 30,

Millipore) and stored at -80 °C. Protein purification of Thermus thermophilus EF-Tu

(pBR322-EF-Tu) was performed essentially as above, but without the incubation step at

60 ºC as previously described (Seidler et al., 1987).

3.2.4. In vitro transcription of tRNA

In vitro T7 RNA polymerase run-off transcription of tRNAPro, tRNALys,

tRNAAla, and tRNALeu was prepared as described (Mursinna et al., 2001). The tRNA

transcript was purified on a denaturing 12 % polyacrylamide gel and recovered by 77 UV-shadowing and subsequent elution from the polyacrylamide gel piece by

electrophoresis. The tRNA was then phenol and chloroform extracted, ethanol

precipitated, and resuspended in DEPC-treated H2O containing 2 mM MgCl2. The

transcribed tRNA was re-folded by incubation for 1 min at 80° C, followed by slow

cooling to 25 °C. In vitro transcribed M. thermautotrophicus tRNALys and tRNAAla

were inactive in aminoacylation (data not shown).

3.2.5. Aminoacylation assays

14 14 L-[U- C]leucine (306 mCi/mmol), L-[U- C]lysine (312 mCi/mmol),

14 14 L-[U- C]proline (241 mCi/mmol), and L-[U- C]alanine (164 mCi/mmol) were all from

Amersham Biosciences. A pre-reaction mixture containing 250 mM KCl, 100 mM

Hepes (pH 7.5), 10 mM dithiothreitol, 10 mM MgCl2, 50 µg/ml BSA, 6 mg/ml

M. thermautotrophicus total tRNA or in vitro transcribed tRNA, and aaRSs at concentrations indicated for specific experiments was pre-incubated for 20 min at room temperature. The appropriate radiolabeled amino acid was then added to the mixture and the temperature increased to 50 °C. After 1 min, 5 mM ATP was added to start the reaction. Aliquots were spotted onto 3MM paper pre-soaked in 5 % TCA (w/v), washed in TCA, and the radioactivity counted.

3.2.6. Preparation of Leu-tRNALeu

To prepare [14C]Leu-tRNALeu, aminoacylation of tRNALeu was performed as above (see section 3.2.5) except that 15 µM L-[14C]leucine (636 cpm/pmol) and

1 µM LeuRS were used. The reaction was incubated for 15 min at 50 ºC and stopped

78 with the addition of 100 mM potassium acetate (pH 4.5) and 250 mM KCl, followed by

phenol/chloroform extraction and ethanol precipitation. The aa-tRNA pellet was dried and resuspended in DEPC-treated water in the presence of 2 mM MgCl2. Radioactivity retained on 3MM filter discs following extensive washing in 5 % TCA to remove unbound radioactive amino acid was counted and the charging level of Leu-tRNALeu

was determined to be ~25 %.

3.2.7. Fluorescence anisotropy experiments: EF-1α with LeuRS and AlaRS

Fluorescent labeling and anisotropy measurements were based upon previously

published procedures (An & Musier-Forsyth, 2005). Briefly, LeuRS, AlaRS, and

EF-1α were labeled with an amine-reactive, extrinsic fluorophore, Alexa Fluor 488

tetrafluorophenyl ester (Molecular Probes, Eugene, OR) at a final molar ratio of

approximately 1:0.8 enzyme:fluorophore. After labeling, proteins were visualized on a

10 % SDS-polyacrylamide gel and subjected to ultraviolet illumination, which

confirmed that the final labeled product contained little or no free fluorophore (data not

shown). Prior to use in fluorescence anisotropy measurements, the activity of

LeuRS-AF, AlaRS-AF, and EF-1α-AF were verified by aminoacylation assays and

protein concentrations determined via active site titration (Fersht et al., 1975). The

concentration of labeled EF-1α-AF was determined by dye binding (BioRad).

Equilibrium dissociation constants were determined by measuring the

fluorescence anisotropy of LeuRS-AF, AlaRS-AF, or EF-1α-AF (100 nM each) as a

function of increasing concentrations of unlabeled protein using a Fluorolog-3

spectrofluorimeter (Horiba Jobin Yvon). The concentration ranges of unlabeled protein 79 used in fluorescence anisotropy experiments with LeuRS-AF and AlaRS-AF were

50-6400 nM unlabeled EF-1α. Alternatively, labeled EF-1α-AF was incubated in the presence of unlabeled AlaRS (50-7000 nM; data not shown). Each protein pair was pre-incubated for 20 min at room temperature in a buffer containing 50 mM Hepes pH

7.5, 250 mM KCl, 10 mM MgCl2, 125 mM glutaric acid, 10 µM GDP, and 5 mM DTT.

All measurements were carried out at least three times and the titration curves were

fitted assuming a 1:1 binding stoichiometry (see section 2.2.7).

3.2.8. Co-immunoprecipitation of EF-1α with LeuRS and AlaRS

Agarose beads coated with Protein A were washed with Hepes, pH 7.5, and

resuspended in 1 ml of the same buffer. Polyclonal antibodies specifically raised

against M. thermautotrophicus EF-1α were bound to the Protein A agarose beads via

shaking at room temperature for 20 min and washed three times in a buffer containing

20 mM Hepes, pH 7.5, 150 mM NaCl, 10 % glycerol, 0.1 % Triton X-100. Anti-EF-1α

antibodies attached to the Protein A agarose beads were resuspended in a buffer

containing Tris-HCl, pH 8.0, 150 mM NaCl, 1 % Triton X-100, 1 mM PMSF; EF-1α

was added, followed by shaking at 4 ºC for 1 h, and the beads then washed two times in

the same buffer. Either labeled LeuRS-AF or AlaRS-AF was then added and the

mixture was shaken at 4 ºC for 1 h. Following incubation, the beads were washed with

900 µl each of buffer 1 (50 mM Hepes (pH 7.5), 500 mM NaCl, 0.2 % Triton X-100,

5 mM EDTA), buffer 2 (50 mM Hepes (pH 7.5), 150 mM NaC1, 0.1 % Triton X-100,

5 mM EDTA, 0.1 % SDS) and buffer 3 (10 mM Tris-HC1 (pH 8.0) and 0.1 % Triton

X-100). The beads were finally resuspended in 20 µl of SDS-PAGE loading buffer and 80 incubated at 100 °C for 5 min. Supernatants were resolved by electrophoresis on a

10 % SDS-PAGE gel and then visualized by fluorescent scanning.

3.2.9. Nucleotide exchange and GTP hydrolysis by EF-1α

EF-1α was assayed for its ability to bind [3H]-GDP retained on nitrocellulose

filters. Nucleotide exchange activity of 0.5 µM EF-1α was measured at 50 ºC in the

presence of 100 µM [3H]-GDP (specific activity 546 cpm/pmol) in a buffer containing

(20 mM Tris, pH 7.5, 50 mM KCl, 10 mM MgCl2, and 1 mM DTT). The exchange reaction was started by the addition of GDP or GTP as previously described (Raimo et

al., 2000). The solution was filtered onto nitrocellulose filters following the reaction,

washed twice with 3 ml cold buffer, dried, and radioactivity counted by liquid

scintillation.

Measurements of the intrinsic GTPase activity of EF-1α were performed in a

32 buffer containing 20 mM Tris (pH 7.5), 10 mM MgCl2, 3 M NaCl, 50 µM [γ- P]GTP

(specific activity 76 cpm/pmol), 1 mM DTT, and 0.4 µM EF-1α in the presence or

absence of LeuRS or BSA at concentrations indicated. Reaction mixtures were

incubated at 50 ºC, and aliquots removed periodically, quenched by addition of ice-cold

32 1 % charcoal (w/v) in 5.6 % perchloric acid, and liberation of [γ- P]Pi measured using

a charcoal adsorption assay on 3MM Whatman filters. The remaining [γ-32P]GTP was

then estimated by scintillation counting.

81 3.2.10. Elongation factor protection of aminoacyl-tRNA against spontaneous

deacylation

M. thermautotrophicus EF-1α was activated in a buffer containing 50 mM Tris

(pH 7.5), 1 mM GTP, 100 mM di-potassium glutarate, 10 mM MgCl2, 25 mM KCl,

5 mM DTT, 1.5 M NH4Cl, 3 mM PEP, and 30 µg/ml pyruvate kinase. T. thermophilus

bacterial EF-Tu was activated in a buffer containing 50 mM Tris (pH 7.5), 70 mM KCl,

0.5 mM GTP, 7 mM MgCl2, 1 mM DTT, 2.5 mM PEP, and 30 µg/ml pyruvate kinase.

To test the protective effects of the archaeal EF-1α or bacterial EF-Tu against

spontaneous hydrolysis of aa-tRNA, [14C]Leu-tRNALeu (0.9 µM) was incubated at

50 ºC in the presence of 4 µM activated EF-α or EF-Tu in activation buffer. At specific time intervals, aliquots were removed, precipitated with cold TCA, filtered on nitrocellulose filters, dried, and radioactivity counted.

3.2.11. Co-purification of LeuRS, LysRS, ProRS, and detection of EF-1α by immunoblotting

M. thermautotrophicus cell-free (S100) extract was applied to a Sephacryl S300

26/60 size-exclusion column (GE Healthcare), equilibrated in buffer B and developed in the same buffer as previously described (see section 2.2.4). Fractions containing the three co-eluting aminoacylation activities of LysRS, LeuRS, and ProRS were pooled

and applied to a second, HiPrep 16/10 Q Sepharose FF anion exchange column

(GE Healthcare) extensively washed and equilibrated in buffer B. The presence of

EF-1α was also analyzed in each eluted fraction from both size-exclusion and anion

exchange chromatography. Proteins were first separated by SDS-PAGE, electroblotted 82 onto nitrocellulose, and each fraction was then assayed for the presence of EF-1α by immunoblotting using polyclonal antibodies specifically raised against

M. thermautotrophicus EF-1α.

3.3. Results

3.3.1. Identification of proteins interacting with EF-1α

An M. thermautotrophicus cDNA library (cloned into the pDEST22 prey

vector) was screened, searching for proteins that interact with archaeal EF-1α. Briefly,

archaeal EF-1α (encoded in the pDBLeu bait vector) was co-transformed in

S. cerevisiae with the M. thermautotrophicus cDNA library, which represents more

than 1000-fold coverage of the archaeal genome. Potentially positive interacting

proteins were identified phenotypically, assaying primarily for the transcriptional

activation of a histidine biosynthesis reporter gene (his3) via growth on selective media

containing 3-aminotriazole (3-AT), a specific inhibitor of the his3 gene product. A

total of 1.4 x 105 transformants were screened for the protein-protein interaction phenotype. Following consistent growth on selective medium containing 25 mM 3-AT, approximately 100 clones were selected for plasmid isolation and sequencing to identify the corresponding genes.

Proteins identified from the screen were divided into four categories based on functional annotation: protein modification, methanogenesis, translation, and those of unknown function (Tables 3.1 and 3.2). Two clones, each identified multiple times, contained genes involved in protein modification: a transglutaminase-like protein

(MTH412) and a chaperonin (MTH218). Both clones have been isolated in previous 83 screens and would not be expected to form specific interactions with EF-1α

(Kasiviswanathan et al., 2006; Praetorius-Ibba et al., 2005; Praetorius-Ibba et al.,

2007). Eight clones, each identified once in the screen, contained genes involved in

methanogenesis (MTH965, MTH1130, MTH1135, MTH1161, MTH1162, MTH1163,

MTH1164, and MTH1165). Methanogenesis protein subunits are known to associate

as higher order complexes and would not be expected to associate with EF-1α

specifically. Additionally, these and similar methanogenic proteins were identified in

previous screens of this M. thermautotrophicus library (Kasiviswanathan et al., 2006;

Praetorius-Ibba et al., 2005; Praetorius-Ibba et al., 2007), suggesting that the

interactions are non-specific.

The third category included an ATP synthase subunit (MTH957), signal

peptidase (MTH1448), magnesium chelatase subunit (MTH673), and three proteins of

unknown function (MTH674, MTH1843, and MTH1858). Similar to the second

category, these proteins were found in previous screens and likely do not form specific

interactions with EF-1α. The fourth category was composed of proteins involved in

translation, LeuRS (MTH1508) and AlaRS (MTH1683), which were each identified

once in the screen. To gain insight into possible links between elongation factors and

other cellular components involved in translation, associations between EF-1α and

LeuRS and AlaRS were further investigated in vitro.

84

r n ). o te n eu) f ted. e d s a r a c og BL i d pe r in d t d i ty n ra anda r v i t t d o pD o n n andar r t ct or andar n I t o t e ; ion s 03 s ctor o t ct v i andar e 2 c t m e t v ion s a s v e

ey t V

r ion s vec t h phe r t c ip t t a e s p c ey a ey r ented as y ion r a w t int M r p e

pr o in e scr s e r t n em t i int rac e bai in l a G e h p g rid t k in p b

De on . u

y ea r oder o insert o int laRS t s S h

in

A M LeuRS N N S W d R n e a d wo-

t pha S. cerevisiae A t Al o s nco t O e e ed p ( nd u a rm Q try α /- + . 5-F + - + o o +/- + +/- + S h r -1

% R

F u .2 E

0 e and n -transf

n + Le

ee A-D (P

co th

uci

f i tw s

n w

be ei

g le th o t α

n o ns

r -1

ow o

i r T

p l A

o th; +++ strong growt lacki

r ract t 3-

). G : EF te r

ow um o M

in con

di

e ions + + m t - /- te gr + - + 5 + ++ ++ vect for ++ + 2 + dia

, terac e lete m odera p

e prey h m

e m -His o

ns between id in v

i t io

in t t br

d th; ++ m y tic c e S h rac d - R e he ow t o la nt gr w A LeuRS nco - - 6 8 es on selec eak s (e for in C t p on sy y DEST

t pP p / /pDEST / o + w dia α α ; e ºC 1 1 h inser en 0 oRS s y h 1. Yeast t r EF- EF- Pr 3 id p 3. owt t h l A l D l B l C ive m t gr o o o o e libra l Leu- Leu- Leu- h a ect asm ow r Pl Contr Contr Contr Contr pDB pDB pDB 48 -, no and Tab sel G

85 Function ORF Description Translation MTH1508 Leucyl-tRNA synthetase MTH1683 Alanyl-tRNA synthetase

Protein Modification * MTH412 Transglutaminase * MTH218 Chaperonin

Methanogenesis MTH965 Tungsten formyl methanofuran dehydrogenase, Subunit F * MTH1130 Methyl coenzyme M reductase II, gamma subunit MTH1135 Methyl viologen-reducing hydrogenase, gamma subunit * MTH1161 N5-methyltetrahydromethanopterin: CoM methyltransferase, C-sub. MTH1162 N5-methyltetrahydromethanopterin: CoM methyltransferase, D-sub. MTH1163 N5-methyltetrahydromethanopterin: CoM methyltransferase, E-sub. MTH1164 Methyl Coenzyme M reductase, alpha subunit * MTH1165 Methyl Coenzyme M reductase, gamma subunit

Other MTH957 ATP synthase subunit C MTH1448 Signal peptidase MTH673 Magnesium chelatase subunit * MTH674 Unknown function MTH1843 Unknown function MTH1858 Unknown function

Table 3.2. Proteins identified as interacting with M. thermautotrophicus EF-1α. *Identified in previous screens (Kasiviswanathan et al., 2006; Praetorius-Ibba et al., 2005; Praetorius-Ibba et al., 2007).

3.3.2. Association of EF-1α with LeuRS and AlaRS

The possible associations of EF-1α with the two aaRSs discovered in the yeast two-hybrid screen, LeuRS and AlaRS, were investigated using antibodies against

EF-1α in co-immunoprecipitation experiments. Briefly, antibody specifically raised against EF-1α was bound and oriented on Protein A agarose beads. Unlabeled EF-1α,

attached to the beads via specific interactions with the antibody against the elongation

factor, was then incubated with fluorescently labeled LeuRS-AF or AlaRS-AF. The

complex was extensively washed, separated on an SDS-PAGE gel, and the presence of

each co-immunoprecipitated aaRS visualized by fluorescence scanning.

86 EF-1α was observed to bind Protein A agarose beads via specific interactions

with its corresponding antibody (Figure 3.1; lane 10), as visualized by Coomassie

Brilliant Blue staining, while the elongation factor was unable to bind in the absence of

antibody (data not shown). LeuRS-AF co-immunoprecipitated specifically with EF-1α

(lane 6), but not in its absence (lane 5), indicating the formation of an EF-1α:LeuRS

complex. Fluorescently labeled AlaRS-AF, however, failed to co-immunoprecipitate

with EF-1α (lane 4), suggesting that EF-1α and AlaRS do not form a stable complex in

vitro under these experimental conditions.

To further investigate protein-protein interactions between EF-1α and the two

aaRSs, anisotropy experiments were employed to determine equilibrium dissociation

constants (KDs) as previously described (see section 2.3.2). The affinity between

LeuRS-AF and EF-1α·GDP was investigated in the presence of increasing concentrations of unlabeled EF-1α, which resulted in a significant increase in

anisotropy. The data were fit to a binding curve assuming a stoichiometry of 1:1, from

which a KD of 730 ± 130 nM was calculated (Figure 3.2A). Although determination of

the binding affinities between each labeled aaRS and EF-1α·GTP would also be of

interest, it is technically impractical to maintain EF-1α in the GTP-bound state under

the conditions required for fluorescence measurements.

87

Figure 3.1. Co-immunoprecipitation of EF-1α with LeuRS and AlaRS. SDS- PAGE visualized by Coomassie Brilliant Blue staining and fluorescence scanning of unlabeled EF-1α with fluorescently labeled AlaRS-AF and LeuRS-AF. Lanes 1 and 9, protein molecular weight markers visualized by fluorescence scanning and Coomassie Brilliant Blue staining, respectively. Lane 2, immunoprecipitation of unlabeled EF-1α. Lanes 2, 3, and 5 show the EF-1α antibody bound by Protein A in the presence of either EF-1α, AlaRS-AF, or LeuRS-AF alone, respectively. Lane 4 shows the co- immunoprecipitation experiment in the presence of EF-1α and AlaRS-AF. Lane 6 shows the co-immunoprecipitation of both LeuRS-AF and EF-1α. Lanes 7 and 8 indicate stock solutions of labeled LeuRS-AF and AlaRS-AF, respectively.

88 The binding of AlaRS-AF to EF-1α could not be detected by fluorescence

anisotropy regardless of which of the two proteins was labeled with the fluorescent dye

(Figure 3.2B). This finding is consistent with the lack of detectable complex formation

between AlaRS-AF and EF-1α in the co-immunoprecipitation experiments. This

suggests that perhaps these two proteins associate only transiently in the cell or that

experimental conditions were not conducive to complex formation; it is also possible

that AlaRS was a false-positive interacting protein of EF-1α in the yeast two-hybrid

screen.

0.250.25 0.250.25

0.200.2 0.200.2

0.15 0.15

py 0.15 0.15 y y y p p p o o ro ro r r ropy t t t t t o s i iso LeuRS-AF· EF-1α iso AlaRS-AF + EF-1α An An iso iso 0.100.1 KD = 730 ± 130 nM 0.100.1 An An

0.050.05 0.050.05

00 0 0 1000 2000 3000 4000 5000 6000 7000 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 [EF-1a] (nM) [EF-1a] (nM) [EF-1α], (µM) [EF-1α], (µM)

Figure 3.2. Fluorescence anisotropy experiments: EF-1α with LeuRS and AlaRS. The binding of AF-labeled LeuRS (A) and AlaRS (B) to unlabeled EF-1α were measured using 100 nM of fluorescently labeled LeuRS-AF or AlaRS-AF, respectively, as a function of increasing concentrations of unlabeled protein. A representative data set is shown, with values shown representing the means and corresponding standard deviations from three independent experiments.

89 3.3.3. Effects of the EF-1α·LeuRS complex on GTP hydrolysis by EF-1α

Efficient protein synthesis requires EF-1α·GTP to bind and deliver aa-tRNAs to

the ribosomal A site during translation elongation. Once codon-anticodon interactions have been established on the ribosome, EF-1α·GTP is hydrolyzed, resulting in the

EF-1α·GDP form. The nucleotide exchange factor then exchanges GDP for GTP,

activating EF-1α for another round of aa-tRNA selection. T. thermophilus EF-Tu, the

bacterial homologue of EF-1α, has been demonstrated to bind and protect the labile

ester bond of certain bacterial aa-tRNAs under specific experimental conditions and is

used as an indirect method to monitor the ability of the elongation factor to bind the aa-

tRNA substrate. T. thermophilus EF-Tu increased the half-life of

M. thermautotrophicus Leu-tRNALeu by almost three-fold, indicating that the bacterial

EF-Tu was able to bind and moderately protect the archaeal aa-tRNA (Figure 3.3).

Although the archaeal EF-1α could be successfully activated with GTP, the protective

effects of EF-1α on the labile M. thermautotrophicus Leu-tRNALeu ester bond were

modest under experimental conditions, providing an approximately two-fold increase in

the half life of the in vitro transcribed aa-tRNA. The relatively low level of aa-tRNA

protection by EF-1α, however, made it impractical to quantify the effects of complex

formation on this particular activity of the elongation factor.

90 Tt EF-Tu

Mth EF-1α

No enzyme

Figure 3.3. Protection of Leu-tRNALeu by archaeal EF-1α and bacterial EF-Tu. Protection of 0.3 µM M. thermautotrophicus [14C]Leu-tRNALeu by 3.0 µM T. thermophilus EF-Tu (■), 3.0 µM M. thermautotrophicus EF-1α (▲), or no enzyme (‹).

As an alternative, the effects of LeuRS on the GTP hydrolysis activity of EF-1α were investigated. EF-1α is known to exist in two forms, GTP- and GDP-bound, where the former species is functionally active to bind aa-tRNA for delivery to the ribosome. The latter form is recycled to the GTP-bound form by a nucleotide exchange factor, producing an EF-1α competent for another round of aa-tRNA selection. As an indirect measurement of this EF-1α function, the intrinsic GTP hydrolysis activity was examined. Due to the lack of observable complex formation between AlaRS and

EF-1α, only the effects of complex formation between EF-1α and LeuRS were

investigated. Upon complex formation with LeuRS, the rate of GTP hydrolysis by

EF-1α was only slightly enhanced as compared to EF-1α alone (Figure 3.4), consistent

with the lack of effects observed during a similar investigation of the human

EF-1α·ValRS complex (Negrutskii et al., 1999). 91

101000

9090 ) )

% 80

( 80 %

s i s is ( y l s o r

d 7070 y H droly P GT

Hy 6060 P GT 5050

4040 1010 220 3030 4040 50 6060 7070 800 990

TimeTime ((minin) )

Figure 3.4. Effects of complex formation on GTP hydrolysis by EF-1α. Measurements of GTP hydrolysis by 0.4 µM EF-1α·[γ-32P]GTP alone (■) were performed in the presence of BSA (▲), or in the presence of a 5-fold (‹) or 10-fold (●) excess of LeuRS. U, no enzyme control.

92 3.3.4. Effects of complex formation between EF-1α and LeuRS on aminoacylation

The potential impact of EF-1α on the activity of LeuRS was monitored with respect to Leu-tRNALeu synthesis. Steady-state aminoacylation kinetics of LeuRS in

the presence or absence of the archaeal EF-1α·GTP indicated that complex formation

specifically enhances the kinetics of tRNALeu aminoacylation, leading to an eight-fold

Leu increase in the kcat for Leu-tRNA synthesis and an overall three-fold enhancement in the rate of aminoacylation by LeuRS (Table 3.3; Figure 3.5 A). This enhancement in leucylation by EF-1α occurred regardless of the guanine nucleotide bound by EF-1α

(i.e. GTP- versus GDP-bound forms) (Figure 3.5 B). Unlike the archaeal EF-1α, the bacterial T. thermophilus EF-Tu had no effect on aminoacylation by LeuRS (Figure 3.5

C), indicating that the enhanced rate of Leu-tRNALeu synthesis was specific to the

archaeal EF-1α and was neither a byproduct of a sequestration event by EF-1α (due to

protection of the labile ester bond leading to an increased population of aa-tRNA) nor

was it the non-specific effect of a GTPase.

1 2 Leu -1 Enzyme Additions KM tRNA (µM) kcat (s ) LeuRS None 1.4 ± 0.03 0.22 ± 0.01 LeuRS EF-1α·GTP 3.8 ± 0.6 1.7 ± 0.02 LeuRS BSA 1.1 ± 0.4 0.34 ± 0.09

Table 3.3. Steady-state aminoacylation kinetics of M. thermautotrophicus LeuRS. 1Enzymes were added at a final concentration of 10 nM. 2Addition of other enzyme components at 3.5 µM.

93 A.

B.

C.

Figure 3.5. Aminoacylation by LeuRS in the presence of archaeal EF-1α and bacterial EF-Tu. (A), Aminoacylation by 10 nM LeuRS alone (■) or in the presence of 3.5 µM M. thermautotrophicus EF-1α·GTP (▲). (B), aminoacylation by 10 nM LeuRS alone (■) in the presence of 3.5 µM EF-1α·GTP (●), EF-1α·GDP (○) or (C), 3.5 µM T. thermophilus EF-Tu·GTP (●) or EF-Tu·GDP (○). ▼, M. thermautotrophicus EF-1α alone.

94 3.3.5. Association of EF-1α with the multi-aminoacyl-tRNA synthetase complex

Formation of a stable complex between EF-1α and LeuRS raised the question

of whether this association is part of the archaeal MSC. In an effort to purify the

archaeal MSC, M. thermautotrophicus cell-free extracts were applied to a Sephacryl

S300 gel filtration column (GE Healthcare) and the aminoacylation activities of LysRS,

LeuRS, and ProRS were assayed in the presence of total archaeal tRNA and the corresponding amino acids as previously described (Figure 2.1 A; see section 2.3.1).

The fractions containing the co-eluted aaRSs were pooled and applied to a second, anion exchange (HiPrep 16/10 Q Sepharose FF, GE Healthcare) column. Again, all three aaRSs co-eluted, suggesting complex formation, while AlaRS displayed a different elution profile consistent with the lack of detectable binding affinities with

EF-1α from co-immunoprecipitation and fluorescence anisotropy experiments (Figure

3.1 and 3.2). Immunoblotting of each eluted fraction using polyclonal antibodies specifically directed against M. thermautotrophicus EF-1α indicated that the elongation factor co-purified with LysRS, LeuRS, and ProRS in fractions from both gel filtration

(S300 26/60, GE Healthcare; Figure 3.6A) and anion exchange (HiPrep 16/10 Q

Sepharose FF, GE Healthcare) chromatography (Figure 3.6B). Further attempts to co-immunoprecipitate EF-1α with LysRS and ProRS, the other two components of the archaeal MSC, were unsuccessful (data not shown), suggesting that EF-1α may associate with the MSC via interactions with LeuRS.

95 A. l) mo p A ( RN t - yl Aminoac

anti-EF1α

B. mol) A (p N -tR yl KCl (M) inoac m A

anti-EF1α

Figure 3.6. Co-purification of LysRS, LeuRS, ProRS, and EF-1α. M. thermautotrophicus cell-free extracts were applied to a Sephacryl S300 gel filtration column and the presence of LysRS, LeuRS, and ProRS was assayed via aminoacylation in the presence of total M. thermautotrophicus tRNA and radiolabeled amino acids (A). Active fractions were then applied to a Q-Sepharose column, extensively washed prior to development with a KCl gradient; the presence of each aaRS was assayed via aminoacylation (B). The presence of EF-1α was assayed in each eluted fraction from gel filtration chromatography displaying co-eluting aaRS aminoacylation activities (A) and in each eluted fraction from anion exchange chromatography (B) by immunoblotting with antibodies against EF-1α (anti-EF-1α). , tRNALys; U, tRNALeu; ▲, tRNAPro; ■, tRNAAla. 96 Previously, we have shown that the association between LeuRS, LysRS, and

ProRS led to an enhancement in the steady-state kinetics of tRNALys and tRNAPro

aminoacylation by three- and five-fold, respectively (Praetorius-Ibba et al., 2005;

Praetorius-Ibba et al., 2007). No enhancement of the catalytic efficiency by LeuRS,

however, was observed when in the presence of LysRS or ProRS. Since EF-1α may

also associate with the archaeal MSC, we explored the effects of complex formation

with EF-1α on the aminoacylation activities of ProRS and LysRS. Unlike the enhanced aminoacylation by LeuRS observed in the presence of EF-1α (Figure 3.7 A), neither

the activities of ProRS nor LysRS were affected by the presence of excess EF-1α in

either the GDP- or GTP-bound forms (Figure 3.7 B and C). Taken together with

previous data, LeuRS enhances the catalytic activities of both LysRS and ProRS, while

aminoacylation by LeuRS is enhanced by EF-1α, resulting in increased aminoacylation of all three associated aaRSs within the archaeal MSC.

97 A. B. +EF-1α·GTP +EF-1α·GDP

mol) mol) (p (p Leu Leu NA NA R

u-tR LeuRS Leu-t Le

1 2 3 4 5 6 7 8 9 Time (min) B. +EF-1α LysRS (pmol) s Ly A N tR s- Ly

0 2 4 6 8 TiTimmee ( m(miinn)) C.

+EF-1α (pmol) o

Pr ProRS Pro-tRNA

0 2 4 6 8 Time (min)

Figure 3.7. Effects of the association of EF-1α and the archaeal multi-aminoacyl- tRNA synthetase complex on the aminoacylation activities of LeuRS, LysRS, and ProRS. Aminoacylation by (A), 10 nM LeuRS (■), (B), 100 nM LysRS (▲) and (C), 80 nM ProRS (‹) alone or in the presence of 3.5 µM EF-1α·GTP (●) or EF-1α·GDP (○). ▼, EF-1α alone.

98 3.4. Discussion

3.4.1. Identification of proteins interacting with archaeal EF-1α

The archaeon M. thermautotrophicus harbors a complex between LeuRS and

EF-1α, a translation elongation factor that specifically binds and escorts aa-tRNAs to

the ribosome. First identified via yeast two-hybrid screening of an

M. thermautotrophicus cDNA library (Table 3.1), complex formation between EF-1α and LeuRS was confirmed by co-immunoprecipitation (Figure 3.1) and fluorescence anisotropy experiments, from which a KD of about 0.7 µM was determined (Figure 3.2).

In complex with EF-1α, aminoacylation by LeuRS was enhanced three-fold, with an

Leu eight-fold increase in the kcat of Leu-tRNA synthesis, suggesting that perhaps a conformational change occurs upon complex formation (Table 3.3). The activities of

EF-1α, however, remained largely unaffected by association with LeuRS.

Unlike the archaeal EF-1α, the bacterial homologue EF-Tu had no effect on aminoacylation by LeuRS, indicating that aminoacylation by LeuRS is specifically enhanced by the archaeal EF-1α and is not an artifact of aa-tRNA protection or a

GTPase activity (Figure 3.5). Archaeal EF-1α enhanced aminoacylation by LeuRS regardless of the bound guanine nucleotide (GDP or GTP), which is in contrast to the

ValRS:EF-1α·GTP complex discovered in H. sapiens, which resulted in enhanced aminoacylation by ValRS only in the presence of EF-1α·GTP (Negrutskii et al., 1999).

This suggests that perhaps the surfaces mediating the interactions between LeuRS with

EF-1α allow the two proteins to remain in contact, regardless of whether aa-tRNA is

bound by EF-1α. This would allow the EF-1α to freely alternate between the

99 GDP-bound enzyme and the GTP-bound form (capable of binding aa-tRNAs), while maintaining interactions with LeuRS. This would also explain the apparent stability of the complex, and its potential role in substrate channeling, since delivery of aa-tRNA to the ribosomal A-site would not necessitate dissociation of LeuRS from EF-1α.

3.4.2. Effects of complex formation on the aminoacylation activities of the archaeal multi-aminoacyl-tRNA synthetase complex

It has been suggested that aminoacylation by class I aaRSs is rate-limited by aa-tRNA release, while class II enzymes are rate-limited by a step prior to product release (Zhang et al., 2006). These distinctions between class I and class II aaRSs may compel an association of translation elongation factors with class I aaRSs for efficient aa-tRNA release while class II aaRSs may not necessarily require stable interactions with EF-1α for product release (Zhang et al., 2006). Stable protein-protein interactions between LeuRS, a class I aaRS, and EF-1α lend support to this notion. AaRSs and EF-

1α perform consecutive functions in translation, aminoacylation of tRNA and subsequent delivery to the ribosome. The association of EF-1α with the archaeal MSC favors the idea of tRNA channeling whereby the aa-tRNA synthesized by the associated aaRSs may be directly handed off to EF-1α for delivery to the ribosome without diffusion (Negrutskii & Deutscher, 1991; Negrutskii & El'skaya, 1998;

Negrutskii et al., 1999).

Whether the LeuRS:EF-1α complex exists as a discrete binary complex and/or instead participates as part of a larger macromolecular machine, is unclear. Preliminary

100 findings suggest that EF-1α is linked to the archaeal MSC, possibly through its interactions with LeuRS. It is, therefore, possible that the EF-1α·LeuRS complex may interact independently or associate with the archaeal MSC, creating a larger complex of translational components. The coupling of EF-1α with LeuRS in a separate complex or as a component of the archaeal MSC could enhance the catalytic efficiency of translation in various ways. Taken together, the association of EF-1α with the archaeal

MSC may contribute to the overall efficiency of translation by enhancing aminoacylation by all three aaRSs, as the catalytic efficiencies of LysRS and ProRS were enhanced when in the presence of LeuRS, while EF-1α has been shown to enhance aminoacylation by LeuRS.

The efficiency of translation may also be enhanced through substrate channeling of aa-tRNA directly from the aaRSs of the archaeal MSC to EF-1α. As EF-1α has been shown to be linked to LysRS, LeuRS, and ProRS through its interactions with

LeuRS, these associations may allow substrate channeling of all three aa-tRNAs synthesized by the aaRSs associated in the archaeal MSC. In conjunction with earlier data, this suggests that complex formation between LeuRS, LysRS, ProRS, and EF-1α may play a role in translation by not only enhancing the catalytic efficiencies of all three aaRSs, but also by providing a mechanism whereby aa-tRNAs synthesized by the archaeal MSC are channeled directly to the ribosome without diffusion into the cytoplasm (Hausmann et al., 2007). EF-1α has also been suggested to associate with the mammalian MSC, shuttling aa-tRNAs synthesized from the associated aaRSs to the ribosome for efficient translation (Guzzo & Yang, 2008; Sang et al., 2002).

101 Complimentary to this finding is the discovery that aaRSs associated with the MSC, such as ArgRS, in mammalian cells provide the ribosome with a pool of aa-tRNAs synthesized specifically for protein synthesis (Kyriacou & Deutscher, 2008).

Mammalian aaRSs have also been demonstrated to co-purify with polysomes, further suggesting coordinated events between aa-tRNA synthesis and delivery to the ribosome

(Popenko et al., 1994). Further studies are now necessary to determine the structure of these MSCs, if they are widespread in archaea, and how they function in translation.

102

CHAPTER 4

STRUCTURAL AND FUNCTIONAL MAPPING OF THE ARCHAEAL

MULTI-AMINOACYL-tRNA SYNTHETASE COMPLEX

4.1. Introduction

The faithful translation of proteins based on a nucleic acid code relies on the correct pairing of amino acid and tRNA molecules and subsequent delivery to the ribosome. AaRSs play an essential role in the synthesis of proteins by accurately attaching the correct amino acid onto the cognate tRNA molecule. Once synthesized, aa-tRNAs are selectively bound by EF-1α as a ternary complex with GTP and ushered to the ribosome during protein synthesis. Although united by this essential task of aminoacylation, aaRSs are divided into two classes (I and II) based on active site topologies (Cusack et al., 1990; Eriani et al., 1990). The active site of class I aaRSs is composed of a Rossmann-nucleotide binding fold, harboring the highly conserved sequence motifs HIGH and KMSKS, while the class II enzymes possess an active site composed of anti-parallel beta sheets. Class I aaRSs are generally monomeric and approach tRNA from the minor groove of the tRNA acceptor stem, aminoacylating the terminal adenosine at the 2’-OH position while class II aaRSs are generally multimeric enzymes that approach the major groove of their respective tRNAs, charging the 3’-OH of the terminal adenosine.

103 The proper pairing of amino acid and cognate tRNA is paramount to the

accurate translation of the genetic code. In order to maintain the fidelity of protein

synthesis and avoid infiltration of the genetic code by non-cognate amino acids,

misactivated amino acids and misacylated tRNAs must be efficiently hydrolyzed. As a

result, some aaRSs have evolved proof-reading mechanisms to specifically recognize and hydrolyze misactivated amino acids (pre-transfer editing), misacylated tRNAs

(post-transfer editing), or both, which may be accomplished at a secondary site (see section 1.3). This can occur at a distinct editing domain appended to the catalytic core of several aaRSs or via a separate trans-editing protein factor. Exemplifying the former, class I LeuRS, ValRS, and IleRS harbor an appended editing domain known as connective peptide 1 (CP1) (Chen et al., 2000; Fukai et al., 2000; Lin et al., 1996;

Silvian et al., 1999). Editing of misacylated tRNAs is thought to occur via a repositioning of the misacylated tRNA CCA-end from the activation site to the editing site located within the appended CP1 domain about 40 Å away (Tukalo et al., 2005).

In some cases when the aaRS does not possess an editing activity against a certain non- cognate amino acid, auxiliary protein factors are recruited to edit misacylated tRNAs in trans. Such is the case for the trans-acting factor YbaK found in bacteria, which stably associates with ProRS to specifically hydrolyze misacylated Cys-tRNAPro, further guarding against the incorporation of non-cognate amino acids into the growing polypeptide chain (An & Musier-Forsyth, 2004; Ruan & Soll, 2005).

Although a few reports of complex formation with additional protein factors have been reported in bacteria, such as the ProRS:YbaK complex, aaRSs are commonly found associated into higher order complexes in eukaryotes and archaea. For example,

104 a higher order MSC composed of nine aaRS activities (aminoacylating cognate lysine, leucine, proline, isoleucine, methionine, glutamine, glutamic acid, asparagine, and aspartic acid) is found in mammalian cells (see section 1.6). Three auxiliary proteins

(p38, p43, and p18) complete the complex, promoting the binding of tRNA and contributing to the stability of the complex. The association of aaRSs and auxiliary proteins to form the macromolecular MSC requires an elaborate network of protein- protein interactions within the complex. For example, genetic and biophysical data suggest that most, if not all, aaRSs within the complex specifically associate with p38 or p43 (or both), which likely enhances the stability of the complex. Several of these protein-protein interactions within the MSC are known to be mediated via N- and

C-terminal appended domains found in the associated eukaryotic aaRSs, which are generally absent from their bacterial and archaeal counterparts (Mirande, 1991); for example, the C-terminal extension of mammalian LeuRS is responsible for association with the mammalian MSC (Ling et al., 2005). These appendages to the catalytic cores of several aaRSs are non-catalytic and instead function to mediate protein-protein interactions or act as general RNA binding domains (Cahuzac et al., 2000; Guigou et al., 2004; Robinson et al., 2000). Conversely, several aaRSs, such as LysRS and

AspRS, associate with the MSC through their catalytic domains, since deletions of the appended domains have no effect on complex formation (Agou & Mirande, 1997;

Francin et al., 2002).

In Methanothermobacter thermautotrophicus, a smaller MSC composed of

LysRS, LeuRS, and ProRS was discovered, the eukaryotic counterparts of which are found associated into the mammalian MSC. Probing the functional association of these

105 translational proteins, it was observed that in complex the aminoacylation efficiencies

of both LysRS and ProRS were enhanced in the presence of LeuRS (see Chapter 2).

Yeast two-hybrid analyses indicated that the archaeal aaRSs associate into a ternary

complex with the possible composition LysRS • (N-)LeuRS(-C) • ProRS (see section

2.3.1). EF-1α was also observed to associate with the archaeal MSC possibly via

specific interactions with LeuRS (Chapter 3). This suggests that LeuRS may act as a

core protein to mediate stable complex formation of the archaeal MSC, since no

homologues of the eukaryotic auxiliary proteins have been found in archaea.

Furthermore, M. thermautotrophicus LeuRS possesses a C-terminal extension beyond

the catalytic core; although shorter than its eukaryotic counterpart, this suggests that

comparable regions of the structurally distinct C-terminus of archaeal LeuRS might

also mediate interactions with other proteins within the MSC. This led us to more

closely investigate the potential role of LeuRS in the archaeal MSC, which revealed an

essential role for this aaRS as a core scaffolding protein within the complex.

4.2. Materials and methods

4.2.1. Protein production and purification of LeuRS mutants

His6 fusion derivatives of N- and C-terminally truncated LeuRS and ∆CP1

LeuRS variants were prepared as previously described (see section 2.2.2). N-terminal

His6 tagged fusion derivatives of LeuRS were prepared by inserting the corresponding

PCR amplified genes into the pET11a expression vector. For the ∆N1 variant, forward primer 5'-CAT ATG CAT CAC CAT CAC CAT CAC GAT AGA TGG AGA GAT

GCT GGC-3' and reverse primer 5'-GGT GGT TGA TCA TTA TTC AAG GTA TAT

106 GGC TGG CT-3' were utilized. For the ∆N2 variant, forward primer

5'-GGT GGT CAT ATG CAT CAC CAT CAC CAT CAC CCT GAT GAC AGA

GAA AAG ATA-3' and reverse primer 5'-GGT GGT TGA TCA TTA TTC AAG GTA

TAT GGC TGG CT-3' were utilized. For the ∆C1 LeuRS variant, forward primer 5'-

GGT GGT CAT ATG CAT CAC CAT CAC CAT CAC GAT ATT GAA AGA AAA

TGG-3' and reverse primer 5'-GGT GGT TGA TCA TTA AAC GGC ATT CAC

AGC CTT GTT-3' were used. For the ∆C2 LeuRS variant, forward primer 5'-GGT

GGT CAT ATG CAT CAC CAT CAC CAT CAC GAT ATT GAA AGA AAA TGG-

3' and reverse primer 5'-GGT GGT TGA TCA TTA TGC ATC ATC TGA GAA GTC

AGG-3' were used. For the ∆C3 variant, forward primer 5'-GGT GGT TGA TCA TTA

GAG GAC TGG CCT ACC ATC CAG-3' and reverse primer 5'-GGT GGT CAT ATG

CAT CAC CAT CAC CAT CAC GAT ATT GAA AGA AAA TGG-3' were used. The

∆CP1 variant was created by PCR amplification of two fragments (5’ and 3’), which were then joined by PCR sewing. A linker domain with the sequence

5’-ACC CTC GAG GAA CAG-3’ was inserted in place of the CP1 sequence. For the

5’ fragment of the ∆CP1 variant, forward primer 5'-GGT GGT CAT ATG CAT CAC

CAT CAC CAT CAC GAT ATT GAA AGA AAA TGG-3' and reverse primer

5'-CTG TTC CTC GAG GGT CTC CTT GAG GGT GAG GGG GTT GCC

ATA AAC-3' were used. For the 3’ fragment of the ∆CP1 variant, forward primer

5'-ACC CTC GAG GAA CAG GAA TTC GCT GAG CGA CCT GTT ATA TGC

CGC T-3' and reverse primer 5'-GGT GGT TGA TCA TTA TTC AAG GTA TAT

GGC TGG CT-3' were used. For the PCR sewing of the two ∆CP1 fragments, forward primer 5'-GGT GGT CAT ATG CAT CAC CAT CAC CAT CAC GAT ATT GAA

107 AGA AAA TGG-3' and reverse primer 5'-GGT GGT TGA TCA TTA TTC AAG GTA

TAT GGC TGG CT-3' were used. Cloning of the LeuRS derivatives was done by

isolating the respective NdeI and BclI fragments and ligating into NdeI and BamHI

digested pET11a.

His6-LeuRS derivatives were produced and purified as previously described.

His6-LeuRS mutants were produced by transforming E. coli BL21-RIL (Stratagene) with each pET11a-LeuRS mutant construct. The resulting transformants were grown

and induced to produce protein using the Overnight Express Auto-induction System 1

(Novagen) following the manufacturer’s protocol. Cell-free extract was produced by

passing cells in buffer A (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 10 mM imidazole) containing a protease inhibitor mixture tablet through a French pressure cell

(Complete Mini, EDTA-free; Roche Applied Science) followed by centrifugation at

70,000 xg for 45 min. The supernatant was then flocculated at 60 °C for 10 min to deplete contaminating E. coli proteins, followed by ultracentrifugation at 100,000 xg

2+ for 1 hour. The supernatant from ultracentrifugation was loaded onto a Ni-NTA

column, washed extensively with buffer A, and eluted with an imidazole gradient

(0-250 mM) in the same buffer. Fractions containing the His6-LeuRS derivatives, as determined by SDS-PAGE Coomassie Brilliant Blue staining were pooled, concentrated by ultrafiltration (Amicon 30, Millipore) and stored at -80 °C.

108 4.2.2. Fluorescent labeling of translational proteins

Equilibrium dissociation constants were determined by measuring the fluorescence anisotropy of a fluorescently labeled translational protein as a function of increasing concentrations of an unlabeled protein as previously described (see section

2.2.6) with the following exceptions. Each of the LeuRS variants and EF-1α were labeled with Alexa Fluor (AF) 488 tetrafluorophenyl ester (Molecular Probes, Eugene,

OR). LeuRS and EF-1α were chosen for fluorescent labeling due to the significantly higher stability they displayed over LysRS and ProRS for the labeling procedure.

LeuRS variants and EF-1α were labeled with AF-488 at a molar ratio of 1:15 enzyme:fluorophore for 20 min at room temperature in a buffer containing 40 mM

Hepes pH 7.5, 50 mM NaCl, and 50 mM KCl. Excess unreacted dye was immediately removed by passage through a 1 ml Sephadex G25 spin column pre-equilibrated in the same buffer. To remove residual free dye, each labeled LeuRS variant or EF-1α sample was dialyzed in buffer A and then applied to a Microcon YM-50 concentrator

(Amicon). The final labeling stoichiometry was determined to be approximately

1:0.4 enzyme:fluorophore by Equation 1,

R = [A494 x (dilution factor)]/[ ε494 x Y) (Eq.1)

Where R is the molar ratio of dye to enzyme (LeuRS variant or EF-1α), A494 represents the absorbance of labeled protein solution at 494 nm, ε494 is the extinction coefficient of the dye, and Y is the final concentration of enzyme-AF determined by active site

109 titration (Fersht et al., 1975). Each fluorescently labeled LeuRS variant and EF-1α sample were visualized on a 10 % SDS-polyacrylamide gel and subjected to ultraviolet illumination, which confirmed that the final labeled product contained little or no free fluorophore. Prior to use in fluorescence anisotropy measurements, the activity of the labeled LeuRS variants and EF-1α were verified by aminoacylation and GDP-exchange assays, respectively, and protein concentrations determined by active site titration and dye binding (BioRad), respectively (data not shown; [(Fersht et al., 1975).

4.2.3. Fluorescence anisotropy experiments

Equilibrium dissociation constants were determined by measuring the fluorescence anisotropy of fluorescently labeled EF-1α or LeuRS variants (50 nM each) as a function of increasing concentrations of unlabeled protein using a Fluorolog-

3 spectrofluorimeter (Horiba Jobin Yvon) as previously described (see section 2.2.7).

The following concentration ranges of unlabeled proteins were used: 0-2000 nM

LysRS, and 0-2400 nM ProRS, full-length LeuRS, ∆CP1 LeuRS, LeuRS C-terminal deletion variants ∆C1 and ∆C2, and LeuRS N-terminal deletion variant ∆N1. All measurements were carried out at least three times. The titration curves were fit to

Equation 2, which assumes a 1:1 binding stoichiometry as previously described (An &

Musier-Forsyth, 2005),

2 1/2 A = Amin + [(Y+S+KD) - {(Y+S+KD) - (4YS)} ] · (Amax - Amin)/(2Y) (Eq. 2)

110 Where A is the measured anisotropy at a particular total concentration of unlabeled

protein, S, and LeuRS-AF (Y), Amin is the minimum anisotropy, Amax is the maximum

anisotropy, and KD is the dissociation constant.

4.2.4. Surface plasmon resonance experiments

The surface plasmon resonance (SPR) experiments were performed utilizing a

Biacore T100 instrument (GE Healthcare). Approximately 2000 resonance units (RU)

of EF-1α ligand protein in 100 mM sodium acetate (pH 4.5) were immobilized to a

carboxymethyl dextran (CM5) chip via primary amine groups according to the

manufacturer’s instructions. As a control, a flow cell was activated and blocked in the

absence of protein, which was then used to subtract RU resulting from non-specific

interactions and the bulk refractive index. Binding experiments were performed at

25 ºC in HBS-EP+ buffer (0.01 M Hepes (pH 7.4), 150 mM NaCl, 3 mM EDTA,

0.05 % v/v Surfactant P20) including 10 mM MgCl2, 50 mM KCl and 100 µM GDP at a flow rate of 10 µl/min. The CM5 chip was regenerated with 30 sec of 10 mM glycine-HCl (pH 2.5) at a flow rate of 25 µl/min. Each analysis cycle consisted of injection of approximately 80 µl of analyte (full-length LeuRS or variant ∆C2) over the

immobilized protein (EF-1α) and the control flow cell, 660 sec dissociation time in

HBS-EP+ buffer, and regeneration of the flow cell for the next round of protein

injection. Final RU values were determined following subtraction of the control from

the protein of interest immobilized to the cell.

111 The Rmax values for protein-protein interactions between the analyte and immobilized protein were calculated according to the manufacturer’s protocol for the

Biacore T100 instrument:

Rmax = (MWA / MWL) x IRU x R (Eq. 3)

where Rmax is the binding capacity of the surface in terms of the response at saturation

(maximum response during injection), MWA and MWL are the molecular weights of the

injected analyte protein and the immobilized ligand protein, IRU is the RU value corresponding to the immobilized protein, and R is the stoichiometric ratio (number of molecules of analyte bound to each molecule of ligand). The dissociation constants were calculated using the average RU under steady-state conditions and data were fitted using the Binding Analysis Biacore T100 Evaluation Software.

4.2.5. Preparation of in vitro transcribed tRNA

In vitro T7 RNA polymerase run-off transcription of M. thermautotrophicus tRNALeu was prepared as described (Mursinna et al., 2001). The tRNA transcript was

purified on a denaturing 12 % polyacrylamide gel and recovered by UV-shadowing and

subsequent elution from the polyacrylamide gel piece by electrophoresis. The tRNA

was then phenol and chloroform extracted, ethanol precipitated, and resuspended in

DEPC H2O containing 2 mM MgCl2. The transcribed tRNA was re-folded by

incubation for 1 min at 80 °C, followed by slow cooling to 25 °C.

112 4.2.6. Activation of EF-1α and bacterial EF-Tu

M. thermautotrophicus EF-1α was activated at 50 ºC for 20 min in a buffer containing 50 mM Tris (pH 7.5), 1 mM GTP, 100 mM di-potassium glutarate, 10 mM

MgCl2, 25 mM KCl, 5 mM DTT, 1.5 M NH4Cl, 3 mM PEP, and 30 µg/ml pyruvate

kinase as previously described (see section 3.2.10). T. thermophilus bacterial EF-Tu

was activated in a buffer containing 50 mM Tris (pH 7.5), 70 mM KCl, 0.5 mM GTP,

7 mM MgCl2, 1 mM DTT, 2.5 mM PEP, and 30 µg/ml pyruvate kinase.

4.2.7. ATP consumption editing assay

As an overall measure of cis-editing, ATP consumption catalyzed by LeuRS was monitored in the presence of non-cognate amino acids. The reaction mixture contained

2 U/ml pyrophosphatase (Roche), 2 or 10 mM leucine, methionine, valine, isoleucine, or norvaline (as indicated in figure legend), 5 µM tRNALeu, 2 mM [ -32P]ATP

(5 cpm/pmol), 0.1 M Na-Hepes (pH 7.2), 30 mM KCl, 10 mM MgCl2, and 1 µM ∆CP1

LeuRS or 1 µM LeuRS in the presence or absence of 5 µM EF-1α or EF-Tu (as indicated in figure legends). The reactions, carried out at 37 ºC, were initiated by the addition of LeuRS. Aliquots were removed at various times and quenched in glacial acetic acid. The remaining [γ-32P]ATP and the [γ-32P]Pi formed during the reaction were separated by TLC on a PEI cellulose plate (Sigma) pre-washed with water. The

TLC was subsequently developed in 0.7 M potassium phosphate (pH 3.5) and the labeled products were visualized and quantified on a Storm Phosphorimager

(Amersham Biosciences). The concentration of Pi (mM) formed during the time-course

113 reaction was calculated by multiplying the measured Pi/ATP ratio by the initial

concentration of ATP (2 mM).

4.3. Results

4.3.1. Defining the roles of LeuRS N- and C-termini on complex formation

The active site of LeuRS, a class I aaRS, is composed of a Rossmann-fold

domain containing two highly conserved sequence motifs: HIGH and KMSKS. These

motifs are located near the N- and C-terminus of LeuRS (Figure 4.1), requiring correct

folding and assembly to create the catalytic core necessary for aminoacylation.

Towards probing the role of LeuRS as a core protein of the archaeal MSC, five LeuRS

variants were created, which were successively truncated from the N-terminus to the

HIGH motif (∆N1 and ∆N2) and from the C-terminus to the KMSKS sequence motif

(∆C1, ∆C2, and ∆C3; Figure 4.1). Truncation variants directly adjacent to the HIGH

and KMSKS motifs at the N- and C-terminus (∆N2 and ∆C3), respectively, were

inactive in aminoacylation and leucine activation (data not shown). With the exception

of truncation ∆N1, all LeuRS variants were inactive in aminoacylation likely due to the

lack of tRNA stabilization via the C-terminus, which is known to associate with the

long variable arm of tRNALeu (data not shown). Although inactive in aminoacylation,

LeuRS variants ∆C1 and ∆C2 were able to correctly bind and activate leucine,

indicating correct protein folding (Table 4.1). Along with LeuRS truncation ∆N1,

LeuRS variants ∆C1 and ∆C2 were utilized to investigate the domains of LeuRS

responsible for mediating protein-protein interactions within the archaeal MSC.

114

7

3 ) ) 1 1 C C ∆ ∆ ( ( 890 890 s, 9 ly us n in al six i a e

n

m

high ons

m ) ) 2 2 C C ∆ ∆ ( ( 794 794 i t o

ar

rmi ter e d n

- l

te w

two

d

) ) 3 3 C C ∆ ∆ ( ( 680 680 De rs

C-

ol 9 o f 6 6

-

b N2), C C

ar ∆

h

3 main in. Sho 1

d S o a 6

S d S

n K m R

u

S e

n do N1 an d a old D L

i M

f 9 ∆ ol 0 . K f 6 e - ting c i ted SC- d 601 nn 601 e en

Doma a na u ) m d 4 ig l 55 ss

seq -fo

(des d Ro

nn s

e

1 (CP1

aci h 91 522

t 4 inu ma

s m

in ptide

mino e th

a i Ros

p N-ter e

CP1).

d w uRS

the

∆ te

ectiv (

Le ) m in 429 n 1 nn a o

ro in i f m a co loca icus CP o

let S n m

∆ o ed

( t de SK

2

D in

2 M ser

a LeuRS

f

in m ing

utotroph Deletio

it 6-4 a d K

do 1 Editing D n m

19 an Ed g a P ants o

y n er ∆

i C h 90

b it H 1 4 t vari 6 . G

n 1 I

ted n M

o

ra i t

a mai 2 e

1

the l fs H

i 1

e f t d CP1 ed d

Do o sep

r d

c o

m o 7

ti

are e 8

-fol h on c

C3), an i ma c n i

nn e ∆ e

4 cat d

4 H qu ma

G Sch

, an y, wh I

. ss

al

1 el

4 d se

H

n

al trun

i C2 ) ) 2 2 N N ∆ ∆ ( ( 20 20 e Ro ons iv u

i

v ∆

t

t m ) ) 1 1 N N ∆ ∆ ( 8 8 ( 8 r c

r

e l e 1 1 nse C1, divid o De ∆ N-te Figure 4.1 c resp in (

115 LeuRS Total protein concentration Active LeuRS concentration Active percentage of variant (BioRad assay) (active site titration) LeuRS variants Full-length 110 µM 20 µM 18 % ∆N1 260 µM 55 µM 21 % ∆N2 200 µM 1 µM < 1% ∆C1 170 µM 20 µM 12 % ∆C2 100 µM 17 µM 17 % ∆C3 5 µM < 0.01 µM < 1 % ∆CP1 190 µM 30 µM 16 %

Table 4.1. Determination of LeuRS concentrations. Total protein concentrations were estimated via the BioRad assay. The concentration of active LeuRS enzyme was determined by the active site titration assay (measurement of leucine activation). The active percentage of LeuRS variants indicates the active proportion of the total protein concentration and provides a means to compare LeuRS variants.

The structural organization of the M. thermautotrohpicus MSC, composed of

LysRS, LeuRS, ProRS, and EF-1α, was investigated in vitro using biophysical approaches. To identify the effects of LeuRS truncation variants on complex formation with the three translational proteins associated within the MSC, pair-wise fluorescence anisotropy experiments were employed to determine equilibrium dissociation constants

(KDs) as previously described (see Chapters 2 and 3). First, the effects of truncating the

extreme N-terminus of LeuRS were investigated; the affinity between full-length

LeuRS or the LeuRS variant ∆N1 and each partner protein was determined in the

presence of increasing concentrations of unlabeled partner protein and the data were fit

to a binding curve assuming a stoichiometry of 1:1 (An & Musier-Forsyth, 2005). Full-

length LeuRS bound LysRS, ProRS, and EF-1α with comparable affinities over a range

of KDs between 80-170 nM (Table 4.2). Deletion of eight amino acid residues from the

116 N-terminus of LeuRS (∆N1 variant) had little effect on the binding of the ProRS and

EF-1α (KDs approximately 110 nM). Deletion of the LeuRS N-terminus had a modest

effect on LysRS binding, with a three-fold increase in binding affinity, suggesting that

the N-terminal region of LeuRS may modulate the LysRS:LeuRS interaction, in agreement with previous yeast two-hybrid analyses. Attempts to investigate the potential impact on binding in more detail were hindered by the lack of viable LeuRS variants further truncated from the N-terminus.

Unlabeled protein Fluorescently labeled KD (nM) protein LysRS Full-length LeuRS 170 ± 40 ProRS Full-length LeuRS 80 ± 10 Full-length LeuRS EF-1α 90 ± 10 LysRS N-terminal deletion (∆N1) 50 ± 10 ProRS N-terminal deletion (∆N1) 110 ± 20 N-terminal deletion (∆N1) EF-1α 110 ± 30

Table 4.2. Fluorescence anisotropy experiments: Full-length LeuRS and LeuRS N-terminal truncations. Binding affinities between full-length or N-terminally truncated LeuRS and LysRS, ProRS, or EF-1α.

The effects of C-terminal LeuRS truncation variants on complex formation with

LysRS, ProRS, and EF-1α were investigated via fluorescence anisotropy. LysRS and

EF-1α each bound truncated LeuRS variants (∆C1 and ∆C2) with affinities (KDs 110 -

160 nM) comparable to that of full-length LeuRS (KDs 90 - 170 nM; Table 4.3).

Truncation of the C-terminus, however, impacted the ability of ProRS to associate with 117 LeuRS (KD >2000 nM) (Table 4.3), indicating complex formation between these two aaRSs is at least partly mediated by the C-terminal extension of LeuRS, again consistent with previous yeast-two hybrid data.

Unlabeled LeuRS variant Fluorescently labeled KD (nM) protein LysRS C-terminal deletion (∆C2) 160 ± 30 ProRS C-terminal deletion (∆C1) > 2000 ProRS C-terminal deletion (∆C2) > 2000 C-terminal deletion (∆C1) EF-1α 150 ± 30 C-terminal deletion (∆C2) EF-1α 110 ± 20

Table 4.3. Fluorescence anisotropy experiments: LeuRS C-terminal truncations. Binding affinities between C-terminally truncated LeuRS and LysRS, ProRS, or EF-1α.

4.3.2. Effects of the LeuRS CP1 editing domain on complex formation.

In addition to the M. thermautotrophicus LeuRS derivatives successively truncated from the N- and C-terminus, the LeuRS editing (CP1) domain was deleted to investigate the effects on complex formation. Located between the two signature catalytic motifs (HIGH and KMSKS) in the primary sequence is an inserted CP1 editing domain. Folding into a distinct globular domain adjacent to the catalytic core, this site separate from the activation domain provides a secondary site able to

specifically recognize and hydrolyze misacylated tRNAs. The ∆CP1 variant was active

in leucine activation (Table 4.1), but inactive in editing of non-cognate amino acids

(see below) and in aminoacylation (data not shown), likely due to a reduction in tRNA

118 stabilization via the CP1 domain. Fluorescence anisotropy experiments were employed

to determine the pair-wise binding affinities between the ∆CP1 LeuRS variant and the

three translational proteins associated in the archaeal MSC. Both LysRS and ProRS

bound the ∆CP1 LeuRS editing mutant (KDs range from 40-110 nM) with affinities

comparable to that of full-length LeuRS (KDs range from 80-170 nM), suggesting that the editing domain is not essential for complex formation (Table 4.4). Deletion of the

LeuRS editing domain, however, had a pronounced effect on the protein-protein interactions with EF-1α, however, resulting in a significant decrease in binding affinity

(KD > 2000 nM) as compared to full-length LeuRS, suggesting that the editing domain

is at least partly involved in mediating protein-protein interactions.

Unlabeled LeuRS variant Fluorescently labeled KD (nM) protein LysRS LeuRS ∆CP1 editing domain 40 ± 10 ProRS LeuRS ∆CP1 editing domain 110 ± 10 LeuRS ∆CP1 editing domain EF-1α > 2000

Table 4.4. Fluorescence anisotropy experiments: Deletion of the LeuRS CP1 editing domain. Binding affinities between LeuRS ∆CP1 editing domain and LysRS, ProRS, or EF-1α.

To more closely investigate the binding kinetics between EF-1α and LeuRS via

the CP1 editing domain, SPR experiments were performed using Biacore T100

instrumentation (GE Healthcare). Briefly, EF-1α was immobilized via amine coupling

to a carboxymethyl dextran (CM5) Biacore chip. For the binding experiments,

119 full-length LeuRS or the ∆CP1 variant were injected, flowing over the immobilized

EF-1α. The data were fitted using the Binding Analysis Biacore T100 Evaluation

Software, assuming a stoichiometry of 1:1. As compared to full-length LeuRS

(KD = 280 ± 60), deletion of the LeuRS CP1 domain significantly impaired interactions

with EF-1α (KD > 2000 nM) (Figure 4.2). Broadly consistent with fluorescence

anisotropy data (Table 4.4), these data suggest that the CP1 editing domain specifically

associates with EF-1α.

In order to avoid misincorporation of non-cognate amino acids into the growing polypeptide chain, LeuRS must efficiently discriminate between cognate leucine and closely related molecules, such as methionine, valine, isoleucine, and norvaline. To do so, LeuRS employs the appended cis-editing CP1 domain to hydrolyze misacylated tRNAs (Tukalo et al., 2005). To probe the effects of complex formation with EF-1α on the editing activities of LeuRS, ATP consumption assays were employed, which provide a means to monitor the overall editing reaction encompassing both the pre- and post-transfer components. The ability of full-length LeuRS to edit the non-cognate substrates methionine, valine, isoleucine, and norvaline was monitored via the ATP consumption assay in both the absence and presence of tRNALeu. Methionine, valine, and isoleucine were not substrates under these experimental conditions, displaying ATP consumption rates comparable to cognate leucine (data not shown).

M. thermautotrophicus LeuRS edited norvaline, however, in a tRNA-dependent manner at significantly higher rates than for cognate leucine (Figure 4.3A). As a negative

control, the ∆CP1 LeuRS variant was shown to be inactive in editing of norvaline,

consistent with deletion of the editing domain (Figure 4.3A). 120

A. RU 400400

s 350 it 350 n

300300 e U

250 ons 250

sp EF-1α • LeuRS e 200200 K = 280 ± 60 nM R D d 150150 ize al 100100 m r

No 5050

00 0 5e-7 1e-6 1.5e-6 2e-6 2.5e-6 3e-6 0 500 1000 15C0on0ce nt ra t io n 2000 2500 3000 [Full-length LeuRS] (nM)

B.

400400

350

its 350

300300 se Un

250 on 250

sp EF-1α • ∆CP1 LeuRS mutant 200200 p Re KD > 2000 nM 150150 ized

100100 rmal

No 5050

0 0 00 502e-07 104e-700 1506e-7 0 20008e-7 251e-600 301.20e-60

[∆CP1 Editing Domain mutant] (nM)

Figure 4.2. Surface plasmon resonance experiments. Affinity of EF-1α binding (A) full-length LeuRS or (B) CP1 editing domain deletion of LeuRS. Shown is a representative data set from the Biacore Affinity Evaluation software.

121 E E: LeuRS + leucine A. F FF:: ddCCP1P1 + norvaline + tRNA 0.6 LeuRS + Nor + tRNALeu

) 0.5 M (m d

e 0.4 m u ns

o 0.3

LeuRS + Nor

ATP c 0.2 LeuRS + Leu + tRNALeu ∆CP1 + Nor + tRNALeu 0.1 2 4 6 8 101214

B B: LeuRS + norvaline + tRNA C TiCme: Le u(RmS i+n le)ucine + tRNA D E D: LeuRS + norvaline + tRNA + EF-1a-GDP B. E: LeuRS + norvaline + tRNA + EF-1a-GTP 0.8 LeuRS + Nor + tRNALeu + EF-1α·GTP

0.7 LeuRS + Nor + tRNALeu ) Leu M LeuRS + Nor + tRNA + EF-1α·GDP 0.6 (m d

e 0.5 m u s

n 0.4 o

0.3 ATP c 0.2 LeuRS + Leu + tRNALeu 0.1 050 5 1100151520 25 B: LeuRS + norvaline + tRNA B C: LeuRS + leucine + tRNA C Time (min) D D: LeuRS + norvaline + tRNA + EF-Tu-GDP C. E E: LeuRS + norvaline + tRNA + EF-Tu-GTP 0.8 LeuRS + Nor + tRNALeu + EF-Tu·GTP Leu 0.7 LeuRS + Nor + tRNA + EF-Tu·GDP LeuRS + Nor + tRNALeu )

M 0.6 m (

d

e 0.5 m

0.4 nsu o

0.3 P c AT 0.2 LeuRS + Leu + tRNALeu 0.1 0 5 10 15 20 25 Time (min)

Figure 4.3. ATP consumption assays. The LeuRS editing reaction was monitored by ATP consumption assays in the presence of (A) cognate leucine (Leu) in the presence of tRNALeu (‹) or non-cognate norvaline (Nor) in the presence (■) or absence (▲) of tRNALeu; tRNA-dependent editing of Nor by the ∆CP1 variant was also assayed (●). (B) tRNA-dependent editing of Nor in the absence (■) or presence of EF-1α·GTP (▼) or EF-1α·GDP (V); tRNA-dependent editing of Leu (‹). (C) tRNA-dependent editing of Nor by LeuRS in the absence (■) or presence of EF-Tu·GTP (▼) or EF- Tu·GDP (V); tRNA-dependent editing of Leu (‹).

122 4.3.3. Effects of LeuRS:EF-1α complex formation on editing by LeuRS

As the CP1 editing domain was shown to form specific interactions with EF-1α,

tRNA-dependent editing by LeuRS in the presence of EF-1α was investigated via ATP consumption assays. Since EF-1α is known to adopt alternative conformations, the overall editing reaction was monitored in the presence of EF-1α·GTP (required for aa-

tRNA binding) and EF-1α·GDP. Regardless of the bound guanine nucleotide, LeuRS

in complex with EF-1α displayed editing activities comparable to that of LeuRS alone

(Figure 4.3B), indicating that EF-1α does not effect the hydrolysis of non-cognate norvaline. Similarly, the bacterial homologue T. thermophilus EF-Tu had no effect on the LeuRS editing reaction regardless of the GTP- or GDP-bound states (Figure 4.3C), consistent with previous data suggesting that EF-Tu does not form specific interactions with M. thermautotrophicus LeuRS to enhance aminoacylation.

4.4. Discussion

4.4.1. Examination of the role LeuRS plays in the formation of the archaeal multi-aminoacyl-tRNA synthetase complex

The archaeal MSC, composed of LysRS, LeuRS, and ProRS was previously discovered via yeast two-hybrid analysis, which indicated the existence of an aaRS ternary complex with the possible composition of LysRS • (N-)LeuRS(-C) • ProRS

(Chapter 2). Interestingly, the eukaryotic counterparts of LysRS, LeuRS, and ProRS associate into a higher order complex with six additional aaRS activities and three auxiliary proteins, p18, p38, and p43. No homologues of the mammalian auxiliary

123 proteins, however, have been observed in archaea. M. thermautotrophicus EF-1α was also found to associate with the archaeal MSC, possibly through specific interactions with LeuRS, suggesting a central role of LeuRS as a platform for complex formation.

Taken together with previous yeast two-hybrid data, this suggests a central role for

LeuRS as a platform for complex formation and assembly in archaea. This led us to more closely investigate the potential role of LeuRS as a core component of the archaeal MSC, probing the domains of LeuRS that mediate protein-protein interactions within the complex.

Towards structural mapping of protein interactions within the MSC, several truncations and domain deletions of the archaeal LeuRS were created, which include truncation mutants from the N- and C-terminus and deletion of the modular editing domain known as CP1. Domain deletions within the catalytic core of LeuRS could not be created, however, due to the positioning of the two catalytic motifs HIGH and

KMSKS near the N- and C-termini, respectively. To monitor the effects of LeuRS truncations and deletion variants on complex formation with LysRS, fluorescence anisotropy experiments were employed. Deletion of the N-terminus of LeuRS had a modest effect on LysRS binding (Table 4.2), suggesting that the N-terminal region of

LeuRS may modulate the LysRS:LeuRS interaction, in agreement with previous yeast two-hybrid analyses. Further attempts to investigate the potential impact on binding were hindered by the lack of viable LeuRS variants truncated from the N-terminus.

When examining the domains of LeuRS that mediate interactions with ProRS, it was discovered that truncation of the LeuRS C-terminus significantly decreased binding to

ProRS, consistent with yeast two-hybrid data (Table 4.3). Further attempts to explore

124 the binding kinetics of the LeuRS:ProRS complex by SPR experiments were unsuccessful, however, likely due to the instability of the ProRS enzyme (data not shown).

Deletion of the LeuRS CP1 editing domain resulted in decreased binding by

EF-1α based on fluorescence anisotropy analyses (Table 4.4) as compared to full-length or LeuRS truncation variants (Tables 4.2 and 4.3). Consistent with fluorescence anisotropy data, deletion of the CP1 editing domain of LeuRS significantly decreased binding to EF-1α in SPR experiments (Table 4.5). Although determination of binding kinetics would be desirable, it was not possible under these experimental conditions, likely due to the instability of proteins over the lengthy association and dissociation phases required for measurements of kinetic parameters in real time (data not shown). Taken together, M. thermautotrophicus LysRS may associate with the N-terminus of LeuRS, which may serve as a platform for complex formation with EF-1α and ProRS, with the proposed composition

EF-1α • (CP1-)LeuRS(-C) • ProRS.

4.4.2. Functional association of LysRS, LeuRS, ProRS, and EF-1α

Previously, the functional consequences of complex formation between LysRS,

LeuRS, ProRS, and EF-1α were probed by determining the steady-state aminoacylation parameters of each aaRS alone or in the archaeal MSC (Chapters 2 and 3). It was discovered that in the presence of LeuRS, the catalytic efficiencies of aminoacylation by LysRS and ProRS were enhanced, while no significant changes in the kinetics of

125 aminoacylation by LeuRS were observed. The addition of EF-1α enhanced

aminoacylation by LeuRS primarily via an increase in the rate of Leu-tRNALeu

synthesis, but had no effect on aminoacylation by LysRS and ProRS. Taken together,

this suggests that the biological role of the archaeal MSC in translation may be to

enhance aminoacylation by the associated aaRSs.

In the present study we find that LysRS and ProRS associate with LeuRS via

the N- and C-terminus, respectively, which may function to stabilize protein-protein

interactions or induce a small conformational change required for enhanced

aminoacylation by LysRS and ProRS. Further attempts to investigate the potential

impact on the functional association between LysRS and LeuRS are limited, however,

due to the inviable nature of the N-terminal truncation mutants beyond deletion of the

terminal eight amino acids. The C-terminus of LeuRS folds into a compact domain

flexibly linked to the catalytic body, which is important for aminoacylation as deletion

of the C-terminal domain of P. horikoshii LeuRS has been shown to abolish

aminoacylation (Fukunaga & Yokoyama, 2005). This is due to the fact that the

C-terminal domain is responsible for recognition of the tRNALeu long variable arm

(Figure 4.4) for proper positioning of the substrate. The binding of LysRS and ProRS to disparate regions of LeuRS may indicate an open complex, which may be essential for the biological role of the archaeal MSC in translation.

126

Figure 4.4. Crystal structure of Pyrococcus horikoshii LeuRS in the aminoacylation state. Model of the P. horikoshii LeuRS:tRNALeu complex in the aminoacylation state. Yellow, tRNALeu; orange, Rossmann-fold domain; red, SC-fold domain; light green, C-terminal domain; cyan, CP1 editing domain. From Fukunaga & Yokoyama, 2005.

127 EF-1α was observed to associate specifically with the CP1 domain of LeuRS;

no alteration in editing by LeuRS was detected, however, in the presence of either

archaeal EF-1α or bacterial EF-Tu. This indicates that EF-1α has no effect on the rate-

limiting step of editing, which is likely to be translocation of the misactivated or

misacylated amino acid from the active site to the editing site. These data also suggest

that EF-1α is unable to abstract misacylated tRNALeu from LeuRS, which would otherwise decrease the ATP consumption rate by depleting tRNALeu. Such a

mechanism is essential for accurate translation, as delivery of misacylated tRNAs to the

ribosome would result in the infiltration of the genetic code by non-cognate amino

acids. These findings are consistent with previous data, which indicated enhanced

synthesis of Leu-tRNALeu could be specifically attributed to complex formation with

EF-1α, while bacterial EF-Tu displayed no effect on aminoacylation or editing by

LeuRS. Taken together with present ATP consumption data, aminoacylation may not

be rate-limiting in the LeuRS editing reaction. This indicates that EF-1α forms specific

interactions with LeuRS which do not affect the overall editing reaction, but instead

enhance the rate of Leu-tRNALeu synthesis, possibly by inducing a small

conformational change in the catalytic body of LeuRS.

EF-1α has previously been shown to enhance aminoacylation by LeuRS

irrespective of the GTP- or GDP-bound state of EF-1α. This suggests that perhaps the

surfaces mediating the interactions between LeuRS with EF-1α allow the two proteins

to remain in contact, regardless of whether aa-tRNA is bound by EF-1α. This would

allow EF-1α to freely alternate between the GDP-bound enzyme and the GTP-bound

128 form (capable of binding aa-tRNAs), while maintaining interactions with LeuRS. This would also explain the apparent stability of the complex, and its potential role in substrate channeling of aa-tRNA, since delivery of aa-tRNA to the ribosomal A-site may not necessitate dissociation of LeuRS from EF-1α. Lending support to this notion of substrate channeling, eukaryotic aaRSs have been observed to co-purify with polysomes, although it remains largely undefined as to whether aaRSs form specific complexes with polysomes or rather associate transiently (Popenko et al., 1994).

The specific complex between LeuRS, a class I aaRS, and EF-1α is in agreement with the suggestion that translation elongation factors may be predisposed to form complexes with class I aaRSs (Negrutskii et al., 1999; Zhang et al., 2006). It has been speculated, however, that the bacterial EF-Tu may be unable to stably associate with certain class I aaRSs that harbor appended CP1 editing domains, which include

ValRS, IleRS, and LeuRS, due to steric clash between CP1 and EF-Tu. In agreement with our current data, EF-Tu plays no apparent role in aminoacylation of cognate amino acids or editing of non-cognate molecules, suggesting that EF-Tu may not form specific interactions with LeuRS. Indeed, based on structural modeling of P. horikoshii

LeuRS:tRNALeu and T. aquifex EF-Tu:tRNAPhe structures, it was observed that the editing domain sterically clashed with EF-Tu (Figure 4.5), consistent with previous predictions that bacterial class I aaRSs harboring an appended CP1 editing domain may not favor an association with EF-Tu.

129

A. B. EF-1α CP1 1 1 S.S.s.s.-1EF- EFα 1α

3 3 2

C. T.aEF. EF-T-Tu u 11

P.h. LeuRS C-terminal 3 Domain 2

Figure 4.5. Structural modeling of a P. horikoshii LeuRS:T. aquifex EF-Tu complex. (A) The crystal structure of the binary P. horikoshii LeuRS:tRNALeu complex (PDB 1WZ2) was superimposed on the structure of T. aquifex EF-Tu:tRNAPhe (PDB 1TUI). The superimposition was performed by overlapping sugar-phosphate backbone atoms of the tRNA at nucleotides 1-16, 33-37, and 67-76 of tRNALeu and tRNAPhe via SwissPDB Viewer (tRNA molecules not shown for effective protein visualization) based on the crystal structure modeling of P. horikoshii LeuRS:tRNALeu and T. aquifex EF-Tu·GDPNP:tRNAPhe. (B) Crystal structure of Sulfolobus solfataricus EF-1α·GDP and (C) T. aquifex EF-Tu·GDPNP:tRNAPhe (tRNA is not shown for clarity).

130 In the present study, however, we find that archaeal EF-1α makes specific

contacts with the LeuRS CP1 editing domain. This contradictory finding may be

explained by the fact that the CP1 editing domain is a distinct globular domain flexibly

linked to the catalytic body (Figure 4.6). Due to this apparent flexibility, the precise

position of the CP1 editing domain has been observed to depend on the orientation of

the tRNA CCA-acceptor end as it enters the editing domain during post-transfer

editing, rotating approximately 20º as observed in crystal structures of P. horikoshii and

up to 35º in T. thermophilus LeuRS. Taken together with the fact that the structure and

domain orientations of EF-1α differ from the bacterial EF-Tu (Figure 4.5), this

reorientation of the CP1 domain may permit the association of EF-1α with the archaeal

LeuRS, resulting in enhanced synthesis of Leu-tRNALeu.

Based on genetic and biophysical experimentation, LeuRS has been implicated to play a central role in mediating protein-protein interactions within the archaeal MSC.

These data suggest that LysRS associates with the N-terminus of LeuRS, which in turn serves as a platform for the association of ProRS and EF-1α with the potential composition EF-1α • (CP1-)LeuRS(-C) • ProRS (Figure 4.7). This potentially open structure would allow for the formation of the heterotetrameric complex archaeal MSC, the biological role of which may serve to enhance aminoacylation of the associated aaRSs and to channel aa-tRNAs directly to the ribosome for efficient protein synthesis in the cell.

131

CP1 Editing domain

C-terminal domain

Figure 4.6. Crystal structure of P. horikoshii. Movement of the CP1 editing domain upon tRNA (yellow) binding. The editing domain rotates by about 20º to avoid steric clash with the 5’ phosphate group of tRNALeu. Magenta spheres, joint residues required for the editing domain reorientation. Black, apo form of P. horikoshii LeuRS where the C-terminal domain is truncated; red, tRNA-bound form of P. horikoshii in the intermediate state prior to the tRNA movement of the CCA-end. Adapted from Fukunaga & Yokoyama, 2005.

132

CP1

EF-1α

LeuRS (core)

LysRS N

C

ProRS ProRS

Figure 4.7. Diagram of the proposed structural and functional association between LysRS, LeuRS, ProRS, and EF-1α. Schematic of the role LeuRS plays in modulating complex formation. Arrows indicate the effects of complex formation leading to enhanced aminoacylation activities of the second partner protein.

133

CHAPTER 5

CONCLUSIONS

Accurate translation of mRNA into protein is essential for cellular viability.

Central to this process are the aaRSs, providing amino acid substrates to the ribosome

in the form of aminoacyl-tRNA. Most evident in eukaryotes, aaRSs are known to

associate into higher order complexes; for example, mammalian cells harbor a multi-

enzyme MSC composed of nine aaRS activities and three auxiliary proteins. Although

the stably associated components have been uncovered, the biological role of this

macromolecular MSC remains largely unknown. To investigate the role aaRS

complexes play in translation outside of the mammalian model system, a systematic

search for proteins interacting with archaeal LysRS and ProRS was undertaken. A

MSC composed of LysRS, LeuRS, and ProRS was identified via yeast two-hybrid

screening of an M. thermautotrophicus cDNA library with LysRS and ProRS as bait proteins (see Chapter 2). Formation of a stable complex between LysRS, LeuRS, and

ProRS was further confirmed in vitro and in vivo via co-purification experiments using both the corresponding His-tagged proteins and archaeal cell-free extracts. Pair-wise binding affinities were determined from fluorescence anisotropy studies, indicating that

LeuRS bound LysRS and ProRS with comparable KDs of about 0.3-0.9 µM, lending support to the formation of a stable MSC in archaea.

134 The functional consequences of complex formation were investigated by

determining the steady-state aminoacylation parameters of each aaRS alone or in the

MSC. In the presence of LeuRS, the catalytic efficiencies of aminoacylation by LysRS

and ProRS were enhanced three- and five-fold, respectively, while no significant

changes in the kinetics of aminoacylation by LeuRS were observed. No further

changes were identified upon addition of the aaRSs in alternative combinations. This

indicates the possible role of an archaeal MSC comprised of three aaRSs in which

LeuRS improves the catalytic efficiencies of tRNA aminoacylation by both LysRS and

ProRS. Interestingly, these three aaRS activities are also found in the macromolecular mammalian MSC, suggesting that the biological role of aaRS complexes in translation may be to enhance aminoacylation by the associated aaRSs.

The archaeon M. thermautotrophicus also harbors a complex between LeuRS and EF-1α, a translation elongation factor that specifically binds and escorts aa-tRNAs to the ribosome (see Chapter 3). First identified via yeast two-hybrid screening of an

M. thermautotrophicus cDNA library, complex formation between EF-1α and LeuRS

was confirmed by co-immunoprecipitation and fluorescence anisotropy experiments,

from which a KD of about 0.7 µM was determined (Hausmann et al., 2007). In complex with EF-1α, aminoacylation by LeuRS was enhanced three-fold, with an

Leu eight-fold increase in the kcat of Leu-tRNA synthesis, suggesting that perhaps a conformational change occurs upon complex formation. The activities of EF-1α, however, remained largely unaffected. Unlike the archaeal EF-1α, the bacterial homologue EF-Tu had no effect on aminoacylation by LeuRS, indicating that the enhanced rate of Leu-tRNALeu synthesis was neither a result of product sequestration 135 by EF-1α (known to protect the labile ester bond of aa-tRNA) nor was it the non- specific effect of a GTPase.

It has been suggested that aminoacylation by class I aaRSs may be rate-limited by aa-tRNA release, while class II enzymes are rate-limited by a step prior to product release (Zhang et al., 2006). These distinctions between class I and class II aaRSs may compel an association of translation elongation factors with class I aaRSs for efficient aa-tRNA release while class II aaRSs may not necessarily require stable interactions with EF-1α for product release. Stable protein-protein interactions between LeuRS, a class I aaRS, and EF-1α lend support to this notion. Further, aaRSs and EF-1α perform consecutive functions in translation, aminoacylation of tRNA and subsequent delivery to the ribosome. Although the molecular mechanism remains largely unknown, complex formation between the elongation factor and an aaRS may favor the direct hand-off of aa-tRNA from the aaRS to the elongation factor without diffusion into the cytoplasm, which was first suggested in the case of the ValRS:EF-1α complex discovered in human cells (Negrutskii & Deutscher, 1991; Negrutskii & El'skaya, 1998;

Negrutskii et al., 1999; Petrushenko et al., 2002; Stapulionis & Deutscher, 1995; Yang et al., 2006).

Based on co-purification and immunoblotting experiments, EF-1α has also been suggested to associate with the M. thermautotrophicus MSC, composed of LysRS,

LeuRS, and ProRS, possibly through interactions with LeuRS (Hausmann et al., 2007).

Leu Although EF-1α enhances the kcat of Leu-tRNA synthesis, no change in aminoacylation by LysRS or ProRS was observed in the presence of EF-1α. In

136 accordance with the proposed substrate channeling model, the association of EF-1α with the archaeal MSC may serve to enhance aminoacylation by the three associated aaRSs and to couple two stages of translation: aminoacylation and subsequent delivery of aa-tRNAs to the ribosome via EF-1α.

Assembly and stability of the mammalian MSC is greatly dependent on the auxiliary proteins p18, p38 and p43 (Kim et al., 2002), homologues of which are not encoded in any archaeal genomes. Yeast two-hybrid data suggested that in archaea

LeuRS may instead maintain the integrity of the complex, and we next focused on defining the regions of LeuRS involved in interactions with LysRS, ProRS and EF-1α.

Based on genetic and biophysical experimentation utilizing LeuRS variants truncated from the N- and C-termini, it was discovered that LysRS may associate with the

N-terminal region of LeuRS, while ProRS associates with the C-terminal domain.

These protein-protein interactions may then, in turn, facilitate enhanced aminoacylation efficiencies of LysRS and ProRS when in complex with LeuRS. Additionally, EF-1α was observed to associate with the CP1 editing domain of LeuRS. Complex formation with EF-1α, however, had no effect on editing by LeuRS regardless of the GTP- or

GDP-bound forms, suggesting that the biological function may instead be to enhance the synthesis of Leu-tRNALeu when in complex with EF-1α. No effect on editing by

LeuRS was observed in the presence of EF-Tu·GTP or EF-Tu·GDP, consistent with previous data indicating that EF-Tu forms no specific interactions with LeuRS and hence has no effect on aminoacylation by LeuRS. Further attempts to investigate the potential impact on the functional association between catalytic core of LeuRS and the

137 three associated translational proteins were limited, however, due to the inviable nature of the N- and C-terminal truncation variants beyond the HIGH and KMSKS motifs.

Therefore, it may be of interest to investigate the structural organization of the archaeal

MSC via X-ray crystallography or cryo-EM studies to examine more closely the domains that mediate protein interactions within the complex.

These data revealed an essential role of LeuRS as a scaffolding protein within the archaeal MSC and suggest a potentially open structure in which LysRS associates with the N-terminus of LeuRS, which in turn serves as a platform for the association of

ProRS and EF-1α with the potential composition EF-1α • (CP1-)LeuRS(-C) • ProRS.

Whereas the mechanisms underlying this effect remain unknown, our findings suggest that direct interactions between aaRSs, as seen extensively in higher eukaryotes, may serve to enhance aminoacylation of the associated aaRSs and to channel aa-tRNAs directly to the ribosome for efficient protein synthesis in the cell. Thus, it is possible that the archaeal and mammalian MSCs share the common feature of improving the efficiency of early steps of translation. Further studies are now necessary to determine if these MSCs are widespread in archaea, which would provide new insights into their possible functions in translation.

Multi-protein complexes harboring aaRSs vary in size from binary to macromolecular. This diversity may indicate that other cellular proteins associate with these known complexes, but have remained undiscovered due to the experimental limitations of purification. It is possible that the true identity of protein components associated with the archaeal MSC in vivo may be masked, possibly due to the fragility of the complex, loosely associated peripheral proteins or transient interactions that

138 disassociate during co-purification procedures. Providing new insight into previously unknown protein factors beyond the easily purified core may be the key to unlocking the biological function of complex formation. Further experimentation will be needed to investigate if additional protein factors interact with the archaeal MSC in vivo to further confer a functional advantage to this essential family of enzymes.

In the present studies, we investigated the functional association of archaeal aaRSs in vitro, discovering an enhancement in the aminoacylation efficiencies of

LysRS, LeuRS, and ProRS when in complex. The physiological role of the archaeal

MSC in vivo, however, remains largely unknown. It will be of interest to investigate if these aaRS complexes associate with polysomes, which would provide evidence for the role of the MSC in substrate channeling of aa-tRNAs. It will also be of interest to study the broader role of the archaeal MSC in vivo. While M. thermautotrophicus is ideal for studies of the complex itself, it has several limitations for studying the broader role of the complex, in particular the lack of a tractable genetic system. Instead, the closely related archaeon Thermococcus kodakaraensis may be employed to study the role of the MSC in vivo (French et al., 2007; Shiraki et al., 2003). The LeuRS (65 % similar),

ProRS (73 %), LysRS (55 %) and EF-1α (76 %) proteins of M. thermautotrophicus and

T. kodakarensis all share a high level of homology over their complete sequences, suggesting that they will also share the ability to form a MSC-type complex. The

T. kodakarensis system may therefore provide a means to test the effects of complex disruption. Furthermore, insight into the subcellular localization of archaeal MSCs and dynamic assays in vivo will provide additional evidence to reveal the physiological role of these multi-enzyme complexes in translation.

139

REFERENCE LIST

Agou F & Mirande M (1997) Aspartyl-tRNA synthetase from rat: in vitro functional analysis of its assembly into the multisynthetase complex. Eur J Biochem 243: 259-267.

Ahn HC, Kim S & Lee BJ (2003) Solution structure and p43 binding of the p38 leucine zipper motif: coiled-coil interactions mediate the association between p38 and p43. FEBS Lett 542: 119-124.

Ambrogelly A, Ahel I, Polycarpo C, Bunjun-Srihari S, Krett B, Jacquin-Becker C, Ruan B, Kohrer C, Stathopoulos C, et al (2002) Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine. J Biol Chem 277: 34749-34754.

An S & Musier-Forsyth K (2004) Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK protein. J Biol Chem 279: 42359-42362.

An S & Musier-Forsyth K (2005) Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex. J Biol Chem 280: 34465-34472.

Andersen GR, Nissen P & Nyborg J (2003) Elongation factors in protein biosynthesis. Trends Biochem Sci 28: 434-441.

Antoun A, Pavlov MY, Lovmar M & Ehrenberg M (2006a) How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis. Mol Cell 23: 183-193.

Antoun A, Pavlov MY, Lovmar M & Ehrenberg M (2006b) How initiation factors tune the rate of initiation of protein synthesis in bacteria. EMBO J 25: 2539-2550.

Aravind L & Koonin EV (2000) Eukaryote-specific domains in translation initiation factors: implications for translation regulation and evolution of the translation system. Genome Res 10: 1172-1184.

Asahara H & Uhlenbeck OC (2005) Predicting the binding affinities of misacylated tRNAs for Thermus thermophilus EF-Tu.GTP. Biochemistry 44: 11254-11261.

Ataide SF & Ibba M (2006) Small molecules: big players in the evolution of protein synthesis. ACS Chem Biol 1: 285-297. 140 Ataide SF, Jester BC, Devine KM & Ibba M (2005) Stationary-phase expression and aminoacylation of a transfer-RNA-like small RNA. EMBO Rep 6: 742-747.

Bailly M, Blaise M, Lorber B, Becker HD & Kern D (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol Cell 28: 228-239.

Bandyopadhyay AK & Deutscher MP (1971) Complex of aminoacyl-transfer RNA synthetases. J Mol Biol 60: 113-122.

Barat C, Datta PP, Raj VS, Sharma MR, Kaji H, Kaji A & Agrawal RK (2007) Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits. Mol Cell 27: 250-261.

Barat C, Lullien V, Schatz O, Keith G, Nugeyre MT, Gruninger-Leitch F, Barre- Sinoussi F, LeGrice SF & Darlix JL (1989) HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J 8: 3279-3285.

Bec G, Kerjan P & Waller JP (1994) Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1 beta gamma delta complex. Essential roles of the NH2-terminal extension of valyl-tRNA synthetase and of the EF-1 delta subunit in complex formation. J Biol Chem 269: 2086-2092.

Becker HD & Kern D (1998) Thermus thermophilus: a link in evolution of the tRNA- dependent amino acid amidation pathways. Proc Natl Acad Sci U S A 95: 12832-12837.

Beebe K, Merriman E & Schimmel P (2003) Structure-specific tRNA determinants for editing a mischarged amino acid. J Biol Chem 278: 45056-45061.

Behrensdorf HA, van de CM, Knies UE, Vandenabeele P & Clauss M (2000) The endothelial monocyte-activating polypeptide II (EMAP II) is a substrate for caspase-7. FEBS Lett 466: 143-147.

Belrhali H, Yaremchuk A, Tukalo M, Berthet-Colominas C, Rasmussen B, Bosecke P, Diat O & Cusack S (1995) The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase. Structure 3: 341-352.

Berthonneau E & Mirande M (2000) A gene fusion event in the evolution of aminoacyl-tRNA synthetases. FEBS Lett 470: 300-304.

Beuning PJ & Musier-Forsyth K (2001) Species-specific differences in amino acid editing by class II prolyl-tRNA synthetase. J Biol Chem 276: 30779-30785.

141 Briza P, Winkler G, Kalchhauser H & Breitenbach M (1986) Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure. J Biol Chem 261: 4288-4294.

Brown JR, Gentry D, Becker JA, Ingraham K, Holmes DJ & Stanhope MJ (2003) Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens. EMBO Rep 4: 692-698.

Buddha MR & Crane BR (2005a) Structure and activity of an aminoacyl-tRNA synthetase that charges tRNA with nitro-tryptophan. Nat Struct Mol Biol 12: 274-275.

Buddha MR & Crane BR (2005b) Structures of tryptophanyl-tRNA synthetase II from Deinococcus radiodurans bound to ATP and tryptophan. Insight into subunit cooperativity and domain motions linked to catalysis. J Biol Chem 280: 31965- 31973.

Buddha MR, Keery KM & Crane BR (2004a) An unusual tryptophanyl tRNA synthetase interacts with nitric oxide synthase in Deinococcus radiodurans. Proc Natl Acad Sci U S A 101: 15881-15886.

Buddha MR, Tao T, Parry RJ & Crane BR (2004b) Regioselective nitration of tryptophan by a complex between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase. J Biol Chem 279: 49567-49570.

Cahuzac B, Berthonneau E, Birlirakis N, Guittet E & Mirande M (2000) A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases. EMBO J 19: 445-452.

Caldas TD, El Yaagoubi A & Richarme G (1998) Chaperone properties of bacterial elongation factor EF-Tu. J Biol Chem 273: 11478-11482.

Calendar R & Berg P (1967) D-Tyrosyl RNA: formation, hydrolysis and utilization for protein synthesis. J Mol Biol 26: 39-54.

Cardoso AM, Polycarpo C, Martins OB & Soll D (2006) A non-discriminating aspartyl-tRNA synthetase from Halobacterium salinarum. RNA Biol 3: 110-114.

Cate JH, Yusupov MM, Yusupova GZ, Earnest TN & Noller HF (1999) X-ray crystal structures of 70S ribosome functional complexes. Science 285: 2095-2104.

Chan B, Weidemaier K, Yip WT, Barbara PF & Musier-Forsyth K (1999) Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription. Proc Natl Acad Sci U S A 96: 459- 464.

142 Chen JF, Guo NN, Li T, Wang ED & Wang YL (2000) CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function. Biochemistry 39: 6726-6731.

Chu S, DeRisi J, Eisen M, Mulholland J, Botstein D, Brown PO & Herskowitz I (1998) The transcriptional program of sporulation in budding yeast. Science 282: 699- 705.

Cirakoglu B, Mirande M & Waller JP (1985) A model for the structural organization of aminoacyl-tRNA synthetases in mammalian cells. FEBS Lett 183: 185-190.

Coluccio A, Bogengruber E, Conrad MN, Dresser ME, Briza P & Neiman AM (2004) Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae. Eukaryot Cell 3: 1464-1475.

Corti O, Hampe C, Koutnikova H, Darios F, Jacquier S, Prigent A, Robinson JC, Pradier L, Ruberg M, et al (2003) The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration. Hum Mol Genet 12: 1427-1437.

Curnow AW, Ibba M & Soll D (1996) tRNA-dependent asparagine formation. Nature 382: 589-590.

Curnow AW, Tumbula DL, Pelaschier JT, Min B & Soll D (1998) Glutamyl- tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc Natl Acad Sci U S A 95: 12838-12843.

Cusack S, Berthet-Colominas C, Hartlein M, Nassar N & Leberman R (1990) A second class of synthetase structure revealed by X-ray analysis of Escherichia coli seryl-tRNA synthetase at 2.5 A. Nature 347: 249-255.

Dagkessamanskaia A, Martin-Yken H, Basmaji F, Briza P & Francois J (2001) Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae. FEMS Microbiol Lett 200: 53-58.

Dale T, Sanderson LE & Uhlenbeck OC (2004) The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid. Biochemistry 43: 6159-6166.

Deinert K, Fasiolo F, Hurt EC & Simos G (2001) Arc1p organizes the yeast aminoacyl- tRNA synthetase complex and stabilizes its interaction with the cognate tRNAs. J Biol Chem 276: 6000-6008.

Dibbelt L & Zachau HG (1981) On the rate limiting step of yeast tRNAPhe aminoacylation. FEBS Lett 129: 173-176.

143 Dobard CW, Briones MS & Chow SA (2007) Molecular mechanisms by which human immunodeficiency virus type 1 integrase stimulates the early steps of reverse transcription. J Virol 81: 10037-10046.

Dock-Bregeon A, Sankaranarayanan R, Romby P, Caillet J, Springer M, Rees B, Francklyn CS, Ehresmann C & Moras D (2000) Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem. Cell 103: 877-884.

Dock-Bregeon AC, Rees B, Torres-Larios A, Bey G, Caillet J & Moras D (2004) Achieving error-free translation; the mechanism of proofreading of threonyl- tRNA synthetase at atomic resolution. Mol Cell 16: 375-386.

Eriani G, Delarue M, Poch O, Gangloff J & Moras D (1990) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347: 203-206.

Faisal W, Symonds P, Panjwani S, Heng Y & Murray JC (2007) Cell-surface associated p43/endothelial-monocyte-activating-polypeptide-II in hepatocellular carcinoma cells induces apoptosis in T-lymphocytes. Asian J Surg 30: 13-22.

Feng L, Sheppard K, Tumbula-Hansen D & Soll D (2005) Gln-tRNAGln formation from Glu-tRNAGln requires cooperation of an asparaginase and a Glu- tRNAGln kinase. J Biol Chem 280: 8150-8155.

Ferri-Fioni ML, Fromant M, Bouin AP, Aubard C, Lazennec C, Plateau P & Blanquet S (2006) Identification in archaea of a novel D-Tyr-tRNATyr deacylase. J Biol Chem 281: 27575-27585.

Fersht AR (1998) Sieves in sequence. Science 280: 541.

Fersht AR, Ashford JS, Bruton CJ, Jakes R, Koch GL & Hartley BS (1975) Active site titration and aminoacyl adenylate binding stoichiometry of aminoacyl-tRNA synthetases. Biochemistry 14: 1-4.

Fersht AR & Dingwall C (1979) Evidence for the double-sieve editing mechanism in protein synthesis. Steric exclusion of isoleucine by valyl-tRNA synthetases. Biochemistry 18: 2627-2631.

Fox PL, Mazumder B, Ehrenwald E & Mukhopadhyay CK (2000) Ceruloplasmin and cardiovascular disease. Free Radic Biol Med 28: 1735-1744.

Francin M, Kaminska M, Kerjan P & Mirande M (2002) The N-terminal domain of mammalian Lysyl-tRNA synthetase is a functional tRNA-binding domain. J Biol Chem 277: 1762-1769.

Fraser CS & Hershey JW (2005) Movement in ribosome translocation. J Biol 4: 8. 144 French SL, Santangelo TJ, Beyer AL & Reeve JN (2007) Transcription and translation are coupled in Archaea. Mol Biol Evol 24: 893-895.

Fukai S, Nureki O, Sekine S, Shimada A, Tao J, Vassylyev DG & Yokoyama S (2000) Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase. Cell 103: 793-803.

Fukunaga R, Fukai S, Ishitani R, Nureki O & Yokoyama S (2004) Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine. J Biol Chem 279: 8396-8402.

Fukunaga R & Yokoyama S (2005) Aminoacylation complex structures of leucyl- tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition. Nat Struct Mol Biol 12: 915-922.

Galani K, Grosshans H, Deinert K, Hurt EC & Simos G (2001) The intracellular location of two aminoacyl-tRNA synthetases depends on complex formation with Arc1p. EMBO J 20: 6889-6898.

Galani K, Hurt E & Simos G (2005) The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism. FEBS Lett 579: 969-975.

Gao W, Goldman E & Jakubowski H (1994) Role of carboxy-terminal region in proofreading function of methionyl-tRNA synthetase in Escherichia coli. Biochemistry 33: 11528-11535.

Gentry DR, Ingraham KA, Stanhope MJ, Rittenhouse S, Jarvest RL, O'Hanlon PJ, Brown JR & Holmes DJ (2003) Variable sensitivity to bacterial methionyl- tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes. Antimicrob Agents Chemother 47: 1784-1789.

Geslain R & Ribas dP (2004) Regulation of RNA function by aminoacylation and editing? Trends Genet 20: 604-610.

Godinic V, Mocibob M, Rocak S, Ibba M & Weygand-Durasevic I (2007) Peroxin Pex21p interacts with the C-terminal noncatalytic domain of yeast seryl-tRNA synthetase and forms a specific ternary complex with tRNA(Ser). FEBS J 274: 2788-2799.

Goldgur Y & Safro M (1994) Aminoacyl-tRNA synthetases from Haloarcula marismortui: an evidence for a multienzyme complex in a procaryotic system. Biochem Mol Biol Int 32: 1075-1083.

145 Golinelli-Cohen MP & Mirande M (2007) Arc1p is required for cytoplasmic confinement of synthetases and tRNA. Mol Cell Biochem 300: 47-59.

Golinelli-Cohen MP, Zakrzewska A & Mirande M (2004) Complementation of yeast Arc1p by the p43 component of the human multisynthetase complex does not require its association with yeast MetRS and GluRS. J Mol Biol 340: 15-27.

Gonen H, Smith CE, Siegel NR, Kahana C, Merrick WC, Chakraburtty K, Schwartz AL & Ciechanover A (1994) Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu. Proc Natl Acad Sci U S A 91: 7648-7652.

Graindorge JS, Senger B, Tritch D, Simos G & Fasiolo F (2005) Role of Arc1p in the modulation of yeast glutamyl-tRNA synthetase activity. Biochemistry 44: 1344- 1352.

Greenberg Y, King M, Kiosses WB, Ewalt K, Yang X, Schimmel P, Reader JS & Tzima E (2007) The novel fragment of tyrosyl tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells. FASEB J .

Gromadski KB, Schummer T, Stromgaard A, Knudsen CR, Kinzy TG & Rodnina MV (2007) Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA. J Biol Chem 282: 35629- 35637.

Grosshans H, Hurt E & Simos G (2000a) An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev 14: 830-840.

Grosshans H, Simos G & Hurt E (2000b) Review: transport of tRNA out of the nucleus-direct channeling to the ribosome? J Struct Biol 129: 288-294.

Guigou L, Shalak V & Mirande M (2004) The tRNA-interacting factor p43 associates with mammalian arginyl-tRNA synthetase but does not modify its tRNA aminoacylation properties. Biochemistry 43: 4592-4600.

Guo F, Cen S, Niu M, Javanbakht H & Kleiman L (2003) Specific inhibition of the synthesis of human lysyl-tRNA synthetase results in decreases in tRNA(Lys) incorporation, tRNA(3)(Lys) annealing to viral RNA, and viral infectivity in human immunodeficiency virus type 1. J Virol 77: 9817-9822.

Guo F, Gabor J, Cen S, Hu K, Mouland AJ & Kleiman L (2005) Inhibition of cellular HIV-1 protease activity by lysyl-tRNA synthetase. J Biol Chem 280: 26018- 26023.

146 Guth EC & Francklyn CS (2007) Kinetic discrimination of tRNA identity by the conserved motif 2 loop of a class II aminoacyl-tRNA synthetase. Mol Cell 25: 531-542.

Guzzo CM & Yang DC (2008) Lysyl-tRNA synthetase interacts with EF1alpha, aspartyl-tRNA synthetase and p38 in vitro. Biochem Biophys Res Commun 365: 718-723.

Halwani R, Cen S, Javanbakht H, Saadatmand J, Kim S, Shiba K & Kleiman L (2004) Cellular distribution of Lysyl-tRNA synthetase and its interaction with Gag during human immunodeficiency virus type 1 assembly. J Virol 78: 7553-7564.

Han JM, Kim JY & Kim S (2003) Molecular network and functional implications of macromolecular tRNA synthetase complex. Biochem Biophys Res Commun 303: 985-993.

Han JM, Lee MJ, Park SG, Lee SH, Razin E, Choi EC & Kim S (2006a) Hierarchical network between the components of the multi-tRNA synthetase complex: implications for complex formation. J Biol Chem 281: 38663-38667.

Han JM, Park SG, Lee Y & Kim S (2006b) Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi- functional protein, p43/AIMP1. Biochem Biophys Res Commun 342: 113-118.

Hansen JL, Schmeing TM, Moore PB & Steitz TA (2002) Structural insights into peptide bond formation. Proc Natl Acad Sci U S A 99: 11670-11675.

Harris CL (1987) An aminoacyl-tRNA synthetase complex in Escherichia coli. J Bacteriol 169: 2718-2723.

Hati S, Ziervogel B, SternJohn J, Wong FC, Nagan MC, Rosen AE, Siliciano PG, Chihade JW & Musier-Forsyth K (2006) Pre-transfer editing by class II prolyl- tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids. J Biol Chem 281: 27862-27872.

Hausmann CD, Praetorius-Ibba M & Ibba M (2007) An aminoacyl-tRNA synthetase:elongation factor complex for substrate channeling in archaeal translation. Nucleic Acids Res 35: 6094-6102.

Henkin TM, Glass BL & Grundy FJ (1992) Analysis of the Bacillus subtilis tyrS gene: conservation of a regulatory sequence in multiple tRNA synthetase genes. J Bacteriol 174: 1299-1306.

Herbert CJ, Labouesse M, Dujardin G & Slonimski PP (1988) The NAM2 proteins from S. cerevisiae and S. douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing. EMBO J 7: 473-483.

147 Horan LH & Noller HF (2007) Intersubunit movement is required for ribosomal translocation. Proc Natl Acad Sci U S A 104: 4881-4885.

Hotokezaka Y, Tobben U, Hotokezaka H, Van Leyen K, Beatrix B, Smith DH, Nakamura T & Wiedmann M (2002) Interaction of the eukaryotic elongation factor 1A with newly synthesized polypeptides. J Biol Chem 277: 18545-18551.

Howard OM, Dong HF, Yang D, Raben N, Nagaraju K, Rosen A, Casciola-Rosen L, Hartlein M, Kron M, et al (2002) Histidyl-tRNA synthetase and asparaginyl- tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells. J Exp Med 196: 781-791.

Ibba M & Soll D (1999) Quality control mechanisms during translation. Science 286: 1893-1897.

Ibba M & Soll D (2004) Aminoacyl-tRNAs: setting the limits of the genetic code. Genes Dev 18: 731-738.

Inagaki Y, Blouin C, Susko E & Roger AJ (2003) Assessing functional divergence in EF-1alpha and its paralogs in eukaryotes and archaebacteria. Nucleic Acids Res 31: 4227-4237.

Ivakhno SS & Kornelyuk AI (2005) Bioinformatic analysis of changes in expression level of tyrosyl-tRNA synthetase during sporulation process in Saccharomyces cerevisiae. Mikrobiol Z 67: 37-49.

Jakubowski H (1990) Proofreading in vivo: editing of homocysteine by methionyl- tRNA synthetase in Escherichia coli. Proc Natl Acad Sci U S A 87: 4504-4508.

Jakubowski H (1996) The synthetic/editing active site of an aminoacyl-tRNA synthetase: evidence for binding of thiols in the editing subsite. Biochemistry 35: 8252-8259.

Jakubowski H & Fersht AR (1981) Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases. Nucleic Acids Res 9: 3105-3117.

Jakubowski H & Goldman E (1984) Quantities of individual aminoacyl-tRNA families and their turnover in Escherichia coli. J Bacteriol 158: 769-776.

Javanbakht H, Halwani R, Cen S, Saadatmand J, Musier-Forsyth K, Gottlinger H & Kleiman L (2003) The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly. J Biol Chem 278: 27644-27651.

Jia J, Arif A, Ray PS & Fox PL (2008) WHEP domains direct noncanonical function of glutamyl-Prolyl tRNA synthetase in translational control of gene expression. Mol Cell 29: 679-690.

148 Kapasi P, Chaudhuri S, Vyas K, Baus D, Komar AA, Fox PL, Merrick WC & Mazumder B (2007) L13a blocks 48S assembly: role of a general initiation factor in mRNA-specific translational control. Mol Cell 25: 113-126.

Kapp LD & Lorsch JR (2004) The molecular mechanics of eukaryotic translation. Annu Rev Biochem 73: 657-704.

Karanasios E, Simader H, Panayotou G, Suck D & Simos G (2007) Molecular determinants of the yeast Arc1p-aminoacyl-tRNA synthetase complex assembly. J Mol Biol 374: 1077-1090.

Karkhanis VA, Boniecki MT, Poruri K & Martinis SA (2006) A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria. J Biol Chem 281: 33217-33225.

Kasiviswanathan R, Shin JH & Kelman Z (2006) DNA binding by the Methanothermobacter thermautotrophicus Cdc6 protein is inhibited by the minichromosome maintenance helicase. J Bacteriol 188: 4577-4580.

Kavran JM & Steitz TA (2007) Structure of the base of the L7/L12 stalk of the Haloarcula marismortui large ribosomal subunit: analysis of L11 movements. J Mol Biol 371: 1047-1059.

Kelley SO, Steinberg SV & Schimmel P (2000) Functional defects of pathogenic human mitochondrial tRNAs related to structural fragility. Nat Struct Biol 7: 862-865.

Kerjan P, Cerini C, Semeriva M & Mirande M (1994) The multienzyme complex containing nine aminoacyl-tRNA synthetases is ubiquitous from Drosophila to mammals. Biochim Biophys Acta 1199: 293-297.

Kern D & Gangloff J (1981) Catalytic mechanism of valyl-tRNA synthetase from baker's yeast. Reaction pathway and rate-determining step in the aminoacylation of tRNAVal. Biochemistry 20: 2065-2074.

Kers JA, Wach MJ, Krasnoff SB, Widom J, Cameron KD, Bukhalid RA, Gibson DM, Crane BR & Loria R (2004) Nitration of a peptide phytotoxin by bacterial nitric oxide synthase. Nature 429: 79-82.

Kim JY, Kang YS, Lee JW, Kim HJ, Ahn YH, Park H, Ko YG & Kim S (2002) p38 is essential for the assembly and stability of macromolecular tRNA synthetase complex: implications for its physiological significance. Proc Natl Acad Sci U S A 99: 7912-7916.

Kim MJ, Park BJ, Kang YS, Kim HJ, Park JH, Kang JW, Lee SW, Han JM, Lee HW, et al (2003) Downregulation of FUSE-binding protein and c-myc by tRNA

149 synthetase cofactor p38 is required for lung cell differentiation. Nat Genet 34: 330-336.

Kim T, Park SG, Kim JE, Seol W, Ko YG & Kim S (2000) Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl- tRNA synthetase complex. J Biol Chem 275: 21768-21772.

Kise Y, Lee SW, Park SG, Fukai S, Sengoku T, Ishii R, Yokoyama S, Kim S & Nureki O (2004) A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase. Nat Struct Mol Biol 11: 149-156.

Kleiman L, Halwani R & Javanbakht H (2004) The selective packaging and annealing of primer tRNALys3 in HIV-1. Curr HIV Res 2: 163-175.

Ko HS, von Coelln R, Sriram SR, Kim SW, Chung KK, Pletnikova O, Troncoso J, Johnson B, Saffary R, et al (2005) Accumulation of the authentic parkin substrate aminoacyl-tRNA synthetase cofactor, p38/JTV-1, leads to catecholaminergic cell death. J Neurosci 25: 7968-7978.

Ko YG, Kim EY, Kim T, Park H, Park HS, Choi EJ & Kim S (2001) Glutamine- dependent antiapoptotic interaction of human glutaminyl-tRNA synthetase with apoptosis signal-regulating kinase 1. J Biol Chem 276: 6030-6036.

Korostelev A & Noller HF (2007) The ribosome in focus: new structures bring new insights. Trends Biochem Sci 32: 434-441.

Kotik-Kogan O, Moor N, Tworowski D & Safro M (2005) Structural basis for discrimination of L-phenylalanine from L-tyrosine by phenylalanyl-tRNA synthetase. Structure 13: 1799-1807.

Kovaleski BJ, Kennedy R, Hong MK, Datta SA, Kleiman L, Rein A & Musier-Forsyth K (2006) In vitro characterization of the interaction between HIV-1 Gag and human lysyl-tRNA synthetase. J Biol Chem 281: 19449-19456.

Kozak M (1999) Initiation of translation in prokaryotes and eukaryotes. Gene 234: 187- 208.

Kudlicki W, Coffman A, Kramer G & Hardesty B (1997) Renaturation of rhodanese by translational elongation factor (EF) Tu. Protein refolding by EF-Tu flexing. J Biol Chem 272: 32206-32210.

Kyriacou SV & Deutscher MP (2008) An important role for the multienzyme aminoacyl-tRNA synthetase complex in mammalian translation and cell growth. Mol Cell 29: 419-427.

150 LaRiviere FJ, Wolfson AD & Uhlenbeck OC (2001) Uniform binding of aminoacyl- tRNAs to elongation factor Tu by thermodynamic compensation. Science 294: 165-168.

Laursen BS, Sorensen HP, Mortensen KK & Sperling-Petersen HU (2005) Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev 69: 101-123.

Lee PC, Bochner BR & Ames BN (1983) Diadenosine 5',5"'-P1,P4-tetraphosphate and related adenylylated nucleotides in Salmonella typhimurium. J Biol Chem 258: 6827-6834.

Lee SW, Cho BH, Park SG & Kim S (2004) Aminoacyl-tRNA synthetase complexes: beyond translation. J Cell Sci 117: 3725-3734.

Levengood J, Ataide SF, Roy H & Ibba M (2004) Divergence in noncognate amino acid recognition between class I and class II lysyl-tRNA synthetases. J Biol Chem 279: 17707-17714.

Levin JG, Guo J, Rouzina I & Musier-Forsyth K (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80: 217-286.

Lim K, Tempczyk A, Bonander N, Toedt J, Howard A, Eisenstein E & Herzberg O (2003) A catalytic mechanism for D-Tyr-tRNATyr deacylase based on the crystal structure of Hemophilus influenzae HI0670. J Biol Chem 278: 13496- 13502.

Lin L, Hale SP & Schimmel P (1996) Aminoacylation error correction. Nature 384: 33- 34.

Lincecum TL, Jr., Tukalo M, Yaremchuk A, Mursinna RS, Williams AM, Sproat BS, Van Den EW, Link A, Van Calenbergh S, et al (2003) Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase. Mol Cell 11: 951-963.

Ling C, Yao YN, Zheng YG, Wei H, Wang L, Wu XF & Wang ED (2005) The C- terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex. J Biol Chem 280: 34755-34763.

Ling J, Roy H & Ibba M (2007) Mechanism of tRNA-dependent editing in translational quality control. Proc Natl Acad Sci U S A 104: 72-77.

Lipman RS, Chen J, Evilia C, Vitseva O & Hou YM (2003) Association of an aminoacyl-tRNA synthetase with a putative metabolic protein in archaea. Biochemistry 42: 7487-7496.

151 Liu HW, Cosa G, Landes CF, Zeng Y, Kovaleski BJ, Mullen DG, Barany G, Musier- Forsyth K & Barbara PF (2005) Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription. Biophys J 89: 3470-3479.

Lopez-Huertas E, Charlton WL, Johnson B, Graham IA & Baker A (2000) Stress induces peroxisome biogenesis genes. EMBO J 19: 6770-6777.

Lovmar M & Ehrenberg M (2006) Rate, accuracy and cost of ribosomes in bacterial cells. Biochimie 88: 951-961.

Lukash TO, Turkivska HV, Negrutskii BS & El'skaya AV (2004) Chaperone-like activity of mammalian elongation factor eEF1A: renaturation of aminoacyl- tRNA synthetases. Int J Biochem Cell Biol 36: 1341-1347.

Mazumder B, Sampath P, Seshadri V, Maitra RK, DiCorleto PE & Fox PL (2003a) Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control. Cell 115: 187-198.

Mazumder B, Seshadri V & Fox PL (2003b) Translational control by the 3'-UTR: the ends specify the means. Trends Biochem Sci 28: 91-98.

McCulley A & Morrow CD (2007) Nucleotides within the anticodon stem are important for optimal use of tRNA(Lys,3) as the primer for HIV-1 reverse transcription. Virology 364: 169-177.

McGuire AT & Mangroo D (2007) Cex1p is a novel cytoplasmic component of the Saccharomyces cerevisiae nuclear tRNA export machinery. EMBO J 26: 288- 300.

Min B, Kitabatake M, Polycarpo C, Pelaschier J, Raczniak G, Ruan B, Kobayashi H, Namgoong S & Soll D (2003) Protein synthesis in Escherichia coli with mischarged tRNA. J Bacteriol 185: 3524-3526.

Minella O, Mulner-Lorillon O, Bec G, Cormier P & Belle R (1998) Multiple phosphorylation sites and quaternary organization of guanine-nucleotide exchange complex of elongation factor-1 (EF-1betagammadelta/ValRS) control the various functions of EF-1alpha. Biosci Rep 18: 119-127.

Mirande M (1991) Aminoacyl-tRNA synthetase family from prokaryotes and eukaryotes: structural domains and their implications. Prog Nucleic Acid Res Mol Biol 40: 95-142.

Mirande M, Gache Y, Le Corre D & Waller JP (1982a) Seven mammalian aminoacyl- tRNA synthetases co-purified as high molecular weight entities are associated within the same complex. EMBO J 1: 733-736.

152 Mirande M, Kellermann O & Waller JP (1982b) Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. II. Structural characterization of the polypeptide components and immunological identification of the methionyl-tRNA synthetase subunit. J Biol Chem 257: 11049-11055.

Mirande M, Lazard M, Martinez R & Latreille MT (1992) Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex. Eur J Biochem 203: 459-466.

Mirande M, Le Corre D & Waller JP (1985) A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl- tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex. Eur J Biochem 147: 281-289.

Mocibob M & Weygand-Durasevic I (2008) The proximal region of a noncatalytic eukaryotic seryl-tRNA synthetase extension is required for protein stability in vitro and in vivo. Arch Biochem Biophys 470: 129-138.

Moore PB & Steitz TA (2003) After the ribosome structures: how does peptidyl transferase work? RNA 9: 155-159.

Morales AJ, Swairjo MA & Schimmel P (1999) Structure-specific tRNA-binding protein from the extreme thermophile Aquifex aeolicus. EMBO J 18: 3475- 3483.

Motorin Y, Wolfson AD, Orlovsky AF & Gladilin KL (1988) Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor. FEBS Lett 238: 262-264.

Mursinna RS, Lee KW, Briggs JM & Martinis SA (2004) Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl- tRNA synthetase. Biochemistry 43: 155-165.

Mursinna RS, Lincecum TL, Jr. & Martinis SA (2001) A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl- tRNALeu. Biochemistry 40: 5376-5381.

Nathanson L & Deutscher MP (2000) Active aminoacyl-tRNA synthetases are present in nuclei as a high molecular weight multienzyme complex. J Biol Chem 275: 31559-31562.

Negrutskii BS & Deutscher MP (1991) Channeling of aminoacyl-tRNA for protein synthesis in vivo. Proc Natl Acad Sci U S A 88: 4991-4995.

Negrutskii BS & Deutscher MP (1992) A sequestered pool of aminoacyl-tRNA in mammalian cells. Proc Natl Acad Sci U S A 89: 3601-3604. 153 Negrutskii BS & El'skaya AV (1998) Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res Mol Biol 60: 47-78.

Negrutskii BS, Shalak VF, Kerjan P, El'skaya AV & Mirande M (1999) Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF- 1alpha in the complex with EF-1H. J Biol Chem 274: 4545-4550.

Nilsson J & Nissen P (2005) Elongation factors on the ribosome. Curr Opin Struct Biol 15: 349-354.

Nissen P, Hansen J, Ban N, Moore PB & Steitz TA (2000) The structural basis of ribosome activity in peptide bond synthesis. Science 289: 920-930.

Nomanbhoy T, Morales AJ, Abraham AT, Vortler CS, Giege R & Schimmel P (2001) Simultaneous binding of two proteins to opposite sides of a single transfer RNA. Nat Struct Biol 8: 344-348.

Norcum MT (1989) Isolation and electron microscopic characterization of the high molecular mass aminoacyl-tRNA synthetase complex from murine erythroleukemia cells. J Biol Chem 264: 15043-15051.

Norcum MT (1991) Structural analysis of the high molecular mass aminoacyl-tRNA synthetase complex. Effects of neutral salts and detergents. J Biol Chem 266: 15398-15405.

Norcum MT (1999) Ultrastructure of the eukaryotic aminoacyl-tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images. FEBS Lett 447: 217-222.

Norcum MT & Warrington JA (1998) Structural analysis of the multienzyme aminoacyl-tRNA synthetase complex: a three-domain model based on reversible chemical crosslinking. Protein Sci 7: 79-87.

Nureki O, Vassylyev DG, Tateno M, Shimada A, Nakama T, Fukai S, Konno M, Hendrickson TL, Schimmel P, et al (1998) Enzyme structure with two catalytic sites for double-sieve selection of substrate. Science 280: 578-582.

Oshikane H, Sheppard K, Fukai S, Nakamura Y, Ishitani R, Numata T, Sherrer RL, Feng L, Schmitt E, et al (2006) Structural basis of RNA-dependent recruitment of glutamine to the genetic code. Science 312: 1950-1954.

Park BJ, Kang JW, Lee SW, Choi SJ, Shin YK, Ahn YH, Choi YH, Choi D, Lee KS, et al (2005a) The haploinsufficient tumor suppressor p18 upregulates p53 via interactions with ATM/ATR. Cell 120: 209-221.

154 Park H, Park SG, Kim J, Ko YG & Kim S (2002) Signaling pathways for TNF production induced by human aminoacyl-tRNA synthetase-associating factor, p43. Cytokine 20: 148-153.

Park SG, Ewalt KL & Kim S (2005b) Functional expansion of aminoacyl-tRNA synthetases and their interacting factors: new perspectives on housekeepers. Trends Biochem Sci 30: 569-574.

Paukstelis PJ, Chen JH, Chase E, Lambowitz AM & Golden BL (2008) Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. Nature 451: 94-97.

Peske F, Rodnina MV & Wintermeyer W (2005) Sequence of steps in ribosome recycling as defined by kinetic analysis. Mol Cell 18: 403-412.

Peske F, Savelsbergh A, Katunin VI, Rodnina MV & Wintermeyer W (2004) Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation. J Mol Biol 343: 1183-1194.

Petrushenko ZM, Budkevich TV, Shalak VF, Negrutskii BS & El'skaya AV (2002) Novel complexes of mammalian translation elongation factor eEF1A.GDP with uncharged tRNA and aminoacyl-tRNA synthetase. Implications for tRNA channeling. Eur J Biochem 269: 4811-4818.

Petrushenko ZM, Negrutskii BS, Ladokhin AS, Budkevich TV, Shalak VF & El'skaya AV (1997) Evidence for the formation of an unusual ternary complex of rabbit liver EF-1alpha with GDP and deacylated tRNA. FEBS Lett 407: 13-17.

Pingoud A, Urbanke C, Krauss G, Peters F & Maass G (1977) Ternary complex formation between elongation factor Tu, GTP and aminoacyl-tRNA: an equilibrium study. Eur J Biochem 78: 403-409.

Polycarpo C, Ambrogelly A, Ruan B, Tumbula-Hansen D, Ataide SF, Ishitani R, Yokoyama S, Nureki O, Ibba M, et al (2003) Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases. Mol Cell 12: 287-294.

Popenko VI, Ivanova JL, Cherny NE, Filonenko VV, Beresten SF, Wolfson AD & Kisselev LL (1994) Compartmentalization of certain components of the protein synthesis apparatus in mammalian cells. Eur J Cell Biol 65: 60-69.

Praetorius-Ibba M, Hausmann CD, Paras M, Rogers TE & Ibba M (2007) Functional association between three archaeal aminoacyl-tRNA synthetases. J Biol Chem 282: 3680-3687.

Praetorius-Ibba M & Ibba M (2003) Aminoacyl-tRNA synthesis in archaea: different but not unique. Mol Microbiol 48: 631-637. 155 Praetorius-Ibba M, Rogers TE, Samson R, Kelman Z & Ibba M (2005) Association between Archaeal prolyl- and leucyl-tRNA synthetases enhances tRNA(Pro) aminoacylation. J Biol Chem 280: 26099-26104.

Putzer H, Gendron N & Grunberg-Manago M (1992) Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence. EMBO J 11: 3117- 3127.

Quevillon S, Agou F, Robinson JC & Mirande M (1997) The p43 component of the mammalian multi-synthetase complex is likely to be the precursor of the endothelial monocyte-activating polypeptide II cytokine. J Biol Chem 272: 32573-32579.

Quevillon S & Mirande M (1996) The p18 component of the multisynthetase complex shares a protein motif with the beta and gamma subunits of eukaryotic elongation factor 1. FEBS Lett 395: 63-67.

Quevillon S, Robinson JC, Berthonneau E, Siatecka M & Mirande M (1999) Macromolecular assemblage of aminoacyl-tRNA synthetases: identification of protein-protein interactions and characterization of a core protein. J Mol Biol 285: 183-195.

Raimo G, Masullo M, Lombardo B & Bocchini V (2000) The archaeal elongation factor 1alpha bound to GTP forms a ternary complex with eubacterial and eukaryal aminoacyl-tRNA. Eur J Biochem 267: 6012-6018.

Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C, Simon S, Lenzen G, Petel F, Wojcik J, et al (2001) The protein-protein interaction map of Helicobacter pylori. Nature 409: 211-215.

Ray PS, Arif A & Fox PL (2007) Macromolecular complexes as depots for releasable regulatory proteins. Trends Biochem Sci 32: 158-164.

Reed VS, Wastney ME & Yang DC (1994) Mechanisms of the transfer of aminoacyl- tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha. J Biol Chem 269: 32932-32936.

Reed VS & Yang DC (1994) Characterization of a novel N-terminal peptide in human aspartyl-tRNA synthetase. Roles in the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha. J Biol Chem 269: 32937-32941.

Rho SB, Kim MJ, Lee JS, Seol W, Motegi H, Kim S & Shiba K (1999) Genetic dissection of protein-protein interactions in multi-tRNA synthetase complex. Proc Natl Acad Sci U S A 96: 4488-4493.

156 Rho SB, Lee JS, Jeong EJ, Kim KS, Kim YG & Kim S (1998) A multifunctional repeated motif is present in human bifunctional tRNA synthetase. J Biol Chem 273: 11267-11273.

Rho SB, Lincecum TL, Jr. & Martinis SA (2002) An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing. EMBO J 21: 6874- 6881.

Robinson JC, Kerjan P & Mirande M (2000) Macromolecular assemblage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein interactions and mechanism of complex assembly. J Mol Biol 304: 983-994.

Rocak S, Landeka I & Weygand-Durasevic I (2002) Identifying Pex21p as a protein that specifically interacts with yeast seryl-tRNA synthetase. FEMS Microbiol Lett 214: 101-106.

Romby P, Caillet J, Ebel C, Sacerdot C, Graffe M, Eyermann F, Brunel C, Moine H, Ehresmann C, et al (1996) The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions. EMBO J 15: 5976-5987.

Roy H & Ibba M (2006) Molecular biology: sticky end in protein synthesis. Nature 443: 41-42.

Roy H & Ibba M (2008) RNA-dependent lipid remodeling by bacterial multiple peptide resistance factors. Proc Natl Acad Sci U S A 105: 4667-4672.

Roy H, Ling J, Irnov M & Ibba M (2004) Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase. EMBO J 23: 4639-4648.

Ruan B & Soll D (2005) The bacterial YbaK protein is a Cys-tRNAPro and Cys-tRNA Cys deacylase. J Biol Chem 280: 25887-25891.

Salazar JC, Ahel I, Orellana O, Tumbula-Hansen D, Krieger R, Daniels L & Soll D (2003) Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates. Proc Natl Acad Sci U S A 100: 13863-13868.

Salazar JC, Zuniga R, Raczniak G, Becker H, Soll D & Orellana O (2001) A dual- specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans. FEBS Lett 500: 129-131.

Sampath P, Mazumder B, Seshadri V, Gerber CA, Chavatte L, Kinter M, Ting SM, Dignam JD, Kim S, et al (2004) Noncanonical function of glutamyl-prolyl- tRNA synthetase: gene-specific silencing of translation. Cell 119: 195-208.

157 Sang LJ, Gyu PS, Park H, Seol W, Lee S & Kim S (2002) Interaction network of human aminoacyl-tRNA synthetases and subunits of elongation factor 1 complex. Biochem Biophys Res Commun 291: 158-164.

Sarkar S, Azad AK & Hopper AK (1999) Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 96: 14366-14371.

Schmidt E & Schimmel P (1995) Residues in a class I tRNA synthetase which determine selectivity of amino acid recognition in the context of tRNA. Biochemistry 34: 11204-11210.

Schon A, Kannangara CG, Gough S & Soll D (1988) Protein biosynthesis in organelles requires misaminoacylation of tRNA. Nature 331: 187-190.

Seidler L, Peter M, Meissner F & Sprinzl M (1987) Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8. Nucleic Acids Res 15: 9263-9277.

Sekine S, Nureki O, Shimada A, Vassylyev DG & Yokoyama S (2001) Structural basis for anticodon recognition by discriminating glutamyl-tRNA synthetase. Nat Struct Biol 8: 203-206.

Senger B, Aphasizhev R, Walter P & Fasiolo F (1995) The presence of a D-stem but not a T-stem is essential for triggering aminoacylation upon anticodon binding in yeast methionine tRNA. J Mol Biol 249: 45-58.

Shalak V, Kaminska M, Mitnacht-Kraus R, Vandenabeele P, Clauss M & Mirande M (2001) The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component. J Biol Chem 276: 23769-23776.

Shiraki K, Tsuji M, Hashimoto Y, Fujimoto K, Fujiwara S, Takagi M & Imanaka T (2003) Genetic, enzymatic, and structural analyses of phenylalanyl-tRNA synthetase from Thermococcus kodakaraensis KOD1. J Biochem 134: 567-574.

Silvian LF, Wang J & Steitz TA (1999) Insights into editing from an ile-tRNA synthetase structure with tRNAile and mupirocin. Science 285: 1074-1077.

Simader H, Hothorn M & Suck D (2006) Structures of the interacting domains from yeast glutamyl-tRNA synthetase and tRNA-aminoacylation and nuclear-export cofactor Arc1p reveal a novel function for an old fold. Acta Crystallogr D Biol Crystallogr 62: 1510-1519.

Simos G, Sauer A, Fasiolo F & Hurt EC (1998) A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases. Mol Cell 1: 235-242.

158 Simos G, Segref A, Fasiolo F, Hellmuth K, Shevchenko A, Mann M & Hurt EC (1996) The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. EMBO J 15: 5437-5448.

Sivaram P & Deutscher MP (1990) Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis. Proc Natl Acad Sci U S A 87: 3665-3669.

Stapulionis R & Deutscher MP (1995) A channeled tRNA cycle during mammalian protein synthesis. Proc Natl Acad Sci U S A 92: 7158-7161.

Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nat Rev Mol Cell Biol 9: 242-253.

Swairjo MA, Morales AJ, Wang CC, Ortiz AR & Schimmel P (2000) Crystal structure of trbp111: a structure-specific tRNA-binding protein. EMBO J 19: 6287-6298.

Takyar S, Hickerson RP & Noller HF (2005) mRNA helicase activity of the ribosome. Cell 120: 49-58.

Torres-Larios A, Dock-Bregeon AC, Romby P, Rees B, Sankaranarayanan R, Caillet J, Springer M, Ehresmann C, Ehresmann B, et al (2002) Structural basis of translational control by Escherichia coli threonyl tRNA synthetase. Nat Struct Biol 9: 343-347.

Tukalo M, Yaremchuk A, Fukunaga R, Yokoyama S & Cusack S (2005) The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post- transfer-editing conformation. Nat Struct Mol Biol 12: 923-930.

Tumbula-Hansen D, Feng L, Toogood H, Stetter KO & Soll D (2002) Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms. J Biol Chem 277: 37184-37190.

Vellekamp G & Deutscher MP (1987) A basic NH2-terminal extension of rat liver arginyl-tRNA synthetase required for its association with high molecular weight complexes. J Biol Chem 262: 9927-9930.

Venema RC, Peters HI & Traugh JA (1991) Phosphorylation of valyl-tRNA synthetase and elongation factor 1 in response to phorbol esters is associated with stimulation of both activities. J Biol Chem 266: 11993-11998.

Williams AM & Martinis SA (2006) Mutational unmasking of a tRNA-dependent pathway for preventing genetic code ambiguity. Proc Natl Acad Sci U S A 103: 3586-3591.

159 Wolfe CL, Warrington JA, Davis S, Green S & Norcum MT (2003) Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII. Protein Sci 12: 2282-2290.

Wolfe CL, Warrington JA, Treadwell L & Norcum MT (2005) A three-dimensional working model of the multienzyme complex of aminoacyl-tRNA synthetases based on electron microscopic placements of tRNA and proteins. J Biol Chem 280: 38870-38878.

Wolfson A & Knight R (2005) Occurrence of the aminoacyl-tRNA synthetases in high- molecular weight complexes correlates with the size of substrate amino acids. FEBS Lett 579: 3467-3472.

Wong FC, Beuning PJ, Silvers C & Musier-Forsyth K (2003) An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing. J Biol Chem 278: 52857-52864.

Xia Q, Hendrickson EL, Zhang Y, Wang T, Taub F, Moore BC, Porat I, Whitman WB, Hackett M, et al (2006) Quantitative proteomics of the archaeon Methanococcus maripaludis validated by microarray analysis and real time PCR. Mol Cell Proteomics 5: 868-881.

Yamane T & Hopfield JJ (1977) Experimental evidence for kinetic proofreading in the aminoacylation of tRNA by synthetase. Proc Natl Acad Sci U S A 74: 2246- 2250.

Yang H, Zheng G, Peng X, Qiang B & Yuan J (2003) D-Amino acids and D-Tyr- tRNA(Tyr) deacylase: stereospecificity of the translation machine revisited. FEBS Lett 552: 95-98.

Yang XL, Otero FJ, Ewalt KL, Liu J, Swairjo MA, Kohrer C, RajBhandary UL, Skene RJ, McRee DE, et al (2006) Two conformations of a crystalline human tRNA synthetase-tRNA complex: implications for protein synthesis. EMBO J 25: 2919-2929.

Zeng Y, Liu HW, Landes CF, Kim YJ, Ma X, Zhu Y, Musier-Forsyth K & Barbara PF (2007) Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer. Proc Natl Acad Sci U S A 104: 12651-12656.

Zhang CM, Perona JJ, Ryu K, Francklyn C & Hou YM (2006) Distinct kinetic mechanisms of the two classes of Aminoacyl-tRNA synthetases. J Mol Biol 361: 300-311.

160