Frameshifting As a Tool in Analysis of Transfer RNA Modification and Translation

Frameshifting as a tool in analysis of transfer RNA modification and translation

Ramunė Leipuvienė

Department of Molecular Biology Umeå University Umeå 2004

Doctoral Dissertation 2004 Department of Molecular Biology Umeå University SE-901 87 Umeå, Sweden

ISBN 91-7305-709-6

Printed by Solfjädern Offset AB Umeå, Sweden

2 CONTENTS

ABSTRACT ...... 5

MAIN PAPERS ...... 7

INTRODUCTION ...... 8

TRANSLATION AND READING FRAME MAINTENANCE ...... 8 Ribosomes ...... 8 Types of translational errors ...... 9 Reading frame maintenance errors ...... 10 Spontaneous frameshifting and cell physiology...... 12 Genetics of translational frame maintenance ...... 12 Programmed frameshifting ...... 13 Ribosomal hopping ...... 15

TRNA PROPERTIES ...... 15 Structure of tRNAs ...... 15 tRNA abundance and codon frequency...... 17 tRNA aminoacylation specificity...... 17 Function of modified nucleosides...... 18

IRON-SULFUR CLUSTERS AND TRNA THIOLATION ...... 19 Iron-sulfur cluster proteins ...... 19 Synthesis of iron-sulfur clusters ...... 21 Other proteins involved in iron-sulfur cluster metabolism...... 23 ISC machinery and tRNA thiolation...... 24

AIMS OF THIS WORK ...... 28

RESULTS AND DISCUSSION...... 29

FRAMESHIFTING INDUCED BY DECREASED STABILITY AND REDUCED

AMINOACYLATION OF A MINOR ARGININE TRNA (PAPER I)...... 30

Arg tRNA 5 The sufF mutant has an altered mnm UCU ...... 30

Arg Arg tRNA 5 tRNA 5 mnm UCU and/or arginyl- mnm UCU levels are decreased in the mutants.....31

Glu tRNA 5 2 Peptidyl- mnm s UUC frameshifts when the ribosome is pausing...... 32

3 RIBOSOME SLIPPAGE INDUCED BY DEFECTS IN THE RIBOSOMAL PROTEIN L9

(PAPER II)...... 33 Frameshift mutation is suppressed by mutations in ribosomal protein L9...... 33 The mechanism of ribosomal slippage induced by L9 mutants is unknown...... 34

ENZYMES REQUIRED FOR TRNA THIOLATION (PAPERS III AND IV)...... 35 Formation of s2C and ms2io6A in tRNA requires functional iron-sulfur proteins ..35 TtcA is involved in thiolation of cytidine at position 32 and may hold an iron- sulfur cluster ...... 37 Lack of IscA specifically decreases the levels of ms2io6A but not that of s2C ...... 37 Two pathways for sulfur in tRNA thiolation ...... 38 tRNA thiolations and frameshifting...... 41

CONCLUSIONS...... 43

ACKNOWLEDGEMENTS ...... 44

REFERENCES ...... 45

PAPERS I-IV

4 ABSTRACT

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene

Arg Arg tRNA 5 tRNA 5 coding for the minor mnm UCU . The altered mnm UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-

Arg tRNA 5 AGA frameshifting site the altered mnm UCU reads the AGA codon

Glu tRNA 5 2 inefficiently. This induces a ribosomal pause, allowing the mnm s UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated.

During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated

5 nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

6 MAIN PAPERS

This thesis contains the following publications referred to by their Roman numerals:

I. Decreased stability and reduced aminoacylation of a minor arginine tRNA mediates ribosomal frameshifting. Ramune Leipuviene and Glenn R. Björk. (2004), Manuscript.

II. Ribosome slippage induced by mutations in the ribosomal protein L9. Ramune Leipuviene and Glenn R. Björk. (2004), Manuscript.

III. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. Ramune Leipuviene, Qiang Qian, and Glenn R. Björk. (2004), Journal of Bacteriology, 186:758-766.

IV. The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. Gunilla Jäger, Ramune Leipuviene, Michael G. Pollard, Qiang Qian, and Glenn R. Björk. (2004), Journal of Bacteriology, 186:750-757.

7 INTRODUCTION

Translation and reading frame maintenance

Ribosomes One of the most important processes in the cell is the conversion of genetic information into proteins. This process is initiated by transcription of information contained in the DNA to messenger RNA (mRNA), which later is translated into proteins. Translation occurs via the synchronized action of ribosomes, mRNA, aminoacylated transfer RNAs, and a huge number of different protein factors.

Large ribonucleoprotein particles termed ribosomes are the main players in translation. The bacterial 70S ribosome consists of small (30S) and large (50S) subunits. The 30S subunit comprises 16S rRNA (1542 nucleotides) and 21 proteins, and the 50S subunit comprises 23S rRNA (2904 nucleotides), 5S rRNA (120 nucleotides), and 36 proteins (Gao et al., 2003). The interface between the subunits is occupied by tRNAs that bind to the A (aminacyl), P (peptidyl), and E (exit) ribosomal binding sites. The A site accepts the incoming aminoacylated tRNA, while the P site holds the tRNA with the nascent peptide chain, and the E site possibly holds the deacylated tRNA before it leaves the ribosome. The anticodons of tRNAs base pair with mRNA codons in the 30S subunit, whereas their acceptor 3´-CCA ends, which carry the growing polypeptide chain or incoming amino acid reach into the 50S subunit. The 50S subunit contains the peptidyl transferase center where the peptide bond formation occurs.

The tRNA binding neighborhoods and subunit interface contact surfaces are dominated by rRNA, indicating that ribosomal function is based on rRNA (Yusupov et al., 2001). 16S rRNA adenosines at positions 1493 and 1492 are responsible for monitoring the correct conformation of the first two base pairs in the codon-anticodon duplex in the A-site, facilitating selection of the correct tRNA to the mRNA codon (Ogle et al., 2003; Ramakrishnan, 2002). During

8 translocation of tRNA, different parts of subunits move as well as whole subunits in respect to each other (Noller et al., 2002). During this process, ribosomal proteins undergo large conformational changes suggesting they have an important role in facilitating the dynamics of translation (Gao et al., 2003).

Types of translational errors As Crick et al. indicated many years ago, translation of genetic information contained in mRNA into an amino acid sequence begins at the starting point and continues without any punctuation by successive reading of adjacent three-base codons (Crick et al., 1961). Most often ribosomes translate mRNA faithfully and produce correct polypeptides. However, occasionally different mistakes arise. A missense error occurs due to inaccurate recruitment of the aminoacyl-tRNA to the ribosomal A-site. Aminoacyl-tRNA is termed non-cognate tRNA when it forms fewer than two canonical base pairs (Watson-Crick or wobble) with the codon and encodes a different amino acid than specified by the codon. Aminoacyl-tRNAs that form three Watson-Crick base pairs with the codon (termed cognate tRNA) or form two Watson-Crick and one wobble base pairs with the codon (termed near-cognate) are expected to bring the correct amino acid for protein synthesis. Missense errors introduce an amino acid substitution in the peptide. Most single amino acid substitutions are not harmful for protein function and occur approximately 3x10-4 per codon (Farabaugh, 2000). Another type of ribosomal translation mistake is processivity errors resulting in non- standard length proteins. If a stop codon is read as a sense codon a longer product will be produced; or polypeptide synthesis is prematurely terminated if either a sense codon is interpreted as a stop codon or the peptidyl-tRNA spontaneously dissociates from the ribosomal P-site (Kurland et al., 1996). Processivity errors also occur when ribosomes switch from the initial reading frame to one of the other two alternative reading frames on the translated mRNA. This brings the ribosome into a sequence coding for an aberrant protein. Often the new reading frame contains a stop codon and the synthesized peptide is released. Unless the truncation is very close to the C terminus, truncated proteins are usually inactive.

Reading frame maintenance errors Ribosomal repositioning occurs when the peptidyl-tRNA detaches from mRNA and pairs either with a suitable overlapping codon (referred to as +1 or -1 frameshifting) or with a non-overlapping codon (referred to as bypassing or hopping) and continues conventional translation. During frameshifting, the tRNA that slips in the P-site can be either an undermodified or otherwise altered cognate tRNA with reduced fitness in the P-site, or a near-cognate normal tRNA forming a weak codon-anticodon interaction (Fig. 1) (Farabaugh and Björk, 1999).

Spontaneous slippage events are very infrequent, probably less than 10-5 per codon (Kurland et al., 1996). The lower frequency of frameshifting errors than the frequency of missense errors is because frameshifting involves a double mistake. The ribosome accepts a non-optimal tRNA in the A-site that subsequently induces a frameshift in the P-site (Farabaugh, 2000; Farabaugh and Björk, 1999).

Frameshifting occurs in competition with normal decoding of the next in-frame codon (Baranov et al., 2004; Farabaugh, 1997). If the translation rate is reduced and the ribosome pauses then even the cognate peptidyl-tRNA can slip on the mRNA (Fig. 2). This may occur when damaged tRNA has difficulty entering the A-site, or when one or another aminoacyl-tRNA is in limited supply. A codon in the ribosomal A-site is termed a ‘hungry’ codon if its cognate aminoacyl-tRNA is depleted. It is well documented that ribosomes stalled at such ‘hungry’ codons frameshift (Paper I; Lindsley and Gallant, 1993; Lindsley et al., 2003; Spanjaard and van Duin, 1988; Stahl et al., 2004; Weiss and Gallant, 1986; Weiss et al., 1988). Depending on the frameshifting site context the shift can occur either towards the left (-1 frameshifting) or right (+1 frameshifting) (Lindsley and

Figure 1. Model of translational frameshifting by near-cognate peptidyl-tRNA or either undermodified or defective cognate peptidyl-tRNA (adapted from (Farabaugh and Björk, 1999)). When such a tRNA enters the ribosomal A-site and thereafter is translocated to the P-site it slips to alternative reading frames on the mRNA.

Figure 2. Model of translational frameshifting and bypassing by cognate peptidyl-tRNA (adapted from (Urbonavicius et al., 2001)). When the tRNA for the A-site codon is mutated, undermodified or depleted from the cell, the ribosome pauses, which in turn induces relocation of peptidyl-tRNA in the P-site.

11 Gallant, 1993). The ability of the peptidyl-tRNA to form at least two base pairs (two Watson-Crick or one Watson-Crick and one wobble) or sometimes even less with the codon in the new frame determines the frameshift direction (Farabaugh and Björk, 1999; Hansen et al., 2003; Matsufuji et al., 1995). When there is no suitable landing codon in the overlapping reading frame, dissociated peptidyl-tRNA·ribosome complex can scan through the mRNA without translation and bypass a number of nucleotides until reaching a cognate codon where the ribosome resumes protein chain elongation immediately downstream of the landing codon (Gallant and Lindsley, 1998; Gallant et al., 2003; Lindsley et al., 2003).

Spontaneous frameshifting and cell physiology Spontaneous frameshifting frequency can be affected by certain physiological conditions (Stahl et al., 2004); e.g. upon entrance into stationary phase frameshifting increases 3- to 6-fold in E. coli (Wenthzel et al., 1998). Changed relative intracellular concentrations of tRNA could be responsible for this increase, since they have been shown to vary at different growth rates (Dong et al., 1996). It has also been observed that both the accumulation of abnormally high concentrations of a particular aminoacyl-tRNA species and the deprivation of other tRNA species increases frameshifting (Kurland et al., 1996).

Sequences directing 1% frameshifting levels are frequent in E. coli genes expressed at a low level. Moderate frameshifting during expression of at least some of those genes is not sufficiently harmful for the cells, since there was no strong evolutionary selection against them (Gurvich et al., 2003).

Genetics of translational frame maintenance Genetic analysis of ribosomal reading frame maintenance is founded on the analysis of suppression of frameshift mutations. Frameshift mutation occurs by addition or deletion of nucleotides in the gene sequence, resulting in an out of frame translation of the mRNA and production of a non-functional protein.

12 Correction of the phenotype is possible if the ribosome shifts back to the original reading frame by a secondary suppression mutation within the same gene, or by a number of different types of extragenic suppressor mutations. The most familiar extragenic frameshift suppressors are mutations in genes coding for tRNA or tRNA modifying enzymes that induce alterations in the tRNA structure. As mentioned earlier, structurally damaged tRNA can cause +1 or -1 frameshifting (Paper I; Bossi and Smith, 1984; O'Mahony et al., 1989; Riddle and Roth, 1972b; Sroga et al., 1992), whilst undermodified tRNA can stimulate +1 frameshifting (Urbonavicius et al., 2001) (Fig.1 and Fig. 2). Mutations in other constitutive parts of the translation machinery also affect the frequency of frameshifting. Mutations in the small ribosomal subunit proteins S4 and S5 increase frameshifting frequency, while S12 reduces the frequency (Atkins et al., 1972). Mutations in the large ribosomal subunit protein L7/L12 (Kirsebom and Isaksson, 1985; Kirsebom et al., 1986) and in segments of 16S and 23S rRNA and translation elongation factor EF-Tu (Kurland et al., 1996) also increase frameshifting frequency. All these elements interact with each other and/or with the aminoacyl-tRNA and influence the specificity and kinetics of the decoding process in the ribosome (Farabaugh, 2000; Kurland et al., 1996; Ogle et al., 2003; Ramakrishnan, 2002; Rodnina et al., 2002).

Programmed frameshifting In standard translation, ribosomes read mRNA without jumping over or retranslating single nucleotides. However, specific mRNAs exist that carry programmed ribosomal frameshifting signals, which increase the probability of tRNA slippage from 1% to 50% in either the +1 or the -1 direction. Two general features of all programmed frameshifting sites are a slippery sequence in the mRNA that favors tRNA movement or misalignment, and a stimulator that enhances the process, probably by induction of a ribosomal pause. The stimulator is a slowly decoded region of mRNA that contains rare or stop codons, forms secondary mRNA structures, or is involved in mRNA-rRNA Shine-Dalgarno-like

Figure 3. Three major classes of functionally important frameshifting (Baranov et al., 2002). Cistrons whose translation results in functional products are grey, and non-functional proteins are dashed. (A) Regulation of the biosynthesis of one product. (B) and (C) Regulation or set a ratio between two products: frameshifting results in longer product (B), or shorter product (C).

interactions (Baranov et al., 2002; Namy et al., 2004). Most programmed frameshifts have a clear purpose. A frameshift site residing early in the gene may regulate the synthesis of a protein (Fig. 3A); whilst a frameshift site in the middle of the gene may either regulate the synthesis of a protein or set a ratio between two functional proteins. The longer frameshift product could bear a distinct function from the standard translation product (Fig. 3B). Alternatively, after frameshifting a truncated product can have modified function to its longer counterpart (Fig. 3C). Genes containing programmed frameshifting sites are compiled in a database (http://recode.genetics.utah.edu/) that includes 85 and 66 genes with +1 and -1 frameshifting sites, respectively (Baranov et al., 2003). Most of the data originates from virus, retrotransposon, and insertion element studies. An example of a prokaryotic +1 programmed frameshifting site is utilized in feedback regulation of the translation of prfB gene coding for release factor 2 (Adamski et al., 1993; Craigen and Caskey, 1986; Persson and Atkins, 1998). An example from eukaryotic cells is antizyme 1 that is expressed after +1 frameshifting, and the frameshifting efficiency is regulated by the levels of polyamines (Ivanov et al., 2000; Matsufuji et al., 1995). Various bacteria use a -1 programmed frameshifting site to set a ratio between gamma and tau subunits of DNA polymerase III (both coded by dnaX) where gamma is the truncated form

14 synthesized after frameshifing (Blinkowa and Walker, 1990; Flower and McHenry, 1990; Larsen et al., 1994). Retroviruses regulate their need for a higher concentration of Gag protein compared to the Gag-Pol protein by low frameshifting levels while translating the gag-pol gene fusion, where pol is in the -1 frame with respect to gag. Most of the ribosomes terminate at the end of the gag gene, but a minority shift to a new frame and continue to polymerize the Pol moiety of the Gag-Pol protein (Jacks and Varmus, 1985; Jacks et al., 1988). Transposons Ty in yeast Saccharomyces cerevisiae also use frameshifting for the synthesis of the same proteins, but frameshifting occurs to +1 frame (Belcourt and Farabaugh, 1990; Farabaugh et al., 1993).

Ribosomal hopping The translation of bacteriophage T4 topoisomerase subunit gene 60 mRNA is the only extensively studied and confirmed example of programmed ribosomal hopping. Gene 60 has two reading frames divided by a 50-nucleotide coding gap. The peptidyl-tRNA detaches from the rRNA, bypasses the gap and resumes translation 3´ of the landing site. The hop over the gap requires five stimulatory signals. They are a nascent peptide coded by the first reading frame, a stop codon immediately following the take-off site, a short stem-loop structure in the beginning of the non-coding gap, and matching take-off and landing sites (Herr et al., 2000, 2004). Changes in any of the stimulatory elements, as well as mutations in the ribosomal protein L9 influence the efficiency of hopping (Herr et al., 2001; Weiss et al., 1990).

tRNA properties

Structure of tRNAs tRNAs are small and stable RNAs containing 74 to 95 nucleotides folded in a cloverleaf secondary structure (Fig. 4). The main tRNA body consists of four major nucleosides (A, U, C, and G). However, nucleosides residing at certain

15 positions are extensively modified (Björk, 1996). Eighty-one of the 96 known modifications present in all RNAs are in tRNA (Rozenski et al., 1999) and 42 are in eubacterial tRNA (Fig. 4). The largest variety of modifications are found in the anticodon wobble position (position 34), and adjacent to and 3’ of the anticodon (position 37). Certain tRNA nucleosides are highly conserved and their base pairing yields an L-shaped tertiary structure (Fig. 4). During translation, the ribosome contacts universally conserved portions of tRNA structures, allowing it to bind different tRNA species in precisely the same way (Yusupov et al., 2001).

Figure 4. tRNA secondary cloverleaf and tertiary L-shaped structures (adapted from (Björk and Hagervall, 2004)). Modified nucleosides found in E. coli and S. enterica tRNAs are indicated. U* is cmnm5U (T. Suzuki, personal communication). Either of modifications mnm5s2U/cmnm5s2U are found in position 34, and either of modifications ms2i6A/ms2io6A are found in position 37 of the specific S. enterica tRNAs. In E. coli tRNAs only ms2i6A is present of ms2i6A/ms2io6A.

16 tRNA abundance and codon frequency E. coli has 79 tRNA genes coding for 46 different amino acid acceptor species (Komine et al., 1990). However, the distribution of these tRNA species is not uniform and when measured at slow growth rate the distribution varies from about 4000 to a couple of hundred molecules per cell (Dong et al., 1996). There are 61 sense codons recognized by tRNAs for 20 amino acids. Therefore, several synonymous codons may code for a single amino acid. Codon usage in genes is not random (Nakamura et al., 2000) and codon frequencies in the mRNA pools correlate with the biased tRNA distribution (Dong et al., 1996; Ikemura, 1981a). Frequently used codons are recognized by major tRNAs and rare codons bind minor tRNAs. In genes encoding abundant proteins, codons corresponding to major tRNAs are used (Ikemura, 1981b) and biased codon usage maximizes the speed and fidelity of translation (Ehrenberg and Kurland, 1984; Ikemura, 1981b). The concentration of minor tRNAs cognate for rare codons is limited, and slow reading of a rare codon in the ribosomal A-site often can cause a translational pause eliciting ribosomal frameshifting or hopping. tRNA aminoacylation specificity Before tRNA can function in translation, it has to be aminoacylated by its specific amino acid activating aminoacyl-tRNA synthetase (aaRS). aaRS recognizes specific tRNAs by similar identity signals in all tRNAs coding for the same amino acid (Giegé et al., 1998). Each tRNA carries a limited number of identity determinants, usually residing in the extremities of the tRNA, the anticodon region, nucleotide 73, and the most distal pairs of the amino acid accepting stem (McClain et al., 1990). However, in a few tRNAs major identity signals are found in the core of the molecule (McClain and Foss, 1988). In addition, several tRNAs contain negative signals which hinder interaction of tRNAs with non-cognate synthetases. Most of the determinants are standard nucleosides (Giegé et al., 1998), although a few cases where the determinants are modified nucleosides also exist.

17 Function of modified nucleosides tRNA nucleoside modifications have several functions including importance in tRNA aminoacylation. For example, E. coli modified nucleosides N-6- threonylcarbamoyladenosine-37 (t6A37), 2-thiouridine-34 (s2U34), and 5- methylaminomethyl-2-thiouridine-34 (mnm5s2U34) are identity elements in tRNAs specific for Ile, Glu, and Lys, respectively (Giegé et al., 1998). The modified nucleoside lysidine-34 (k2C34) functions as a positive determinant of tRNAIle for IleRS but it also acts as a negative determinant hindering mis- aminoacylation by MetRS (Muramatsu et al., 1988). Another critical function of modified nucleosides is in modulation of translational efficiency. Modifications in the anticodon wobble position (position 34), which base pair with the third nucleotide of the codon can either extend or restrict the decoding capacity of tRNAs (Björk, 1996). Modifications extending the wobble capacity are uridine 5-oxyacetic acid (cmo5U) and uridine 5- oxyacetic acid methyl ester (mcmo5U) that according to in vivo data allow base pairing with XXA, XXG, XXU, and with codons ending with C for at least proline and alanine tRNAs (Gabriel et al., 1996; Näsvall et al., 2004). Modifications 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5- methylaminomethyl-2´-O-methyluridine (mnm5Um), and 5- methylaminomethyluridine (mnm5U) induce the opposite effect and only allow recognition of codons XXA and XXG (though less efficiently). Lysylation of C34 eliminates recognition of AUG and completely converts the codon specificity as it only allows base pairing to the AUA codon (Muramatsu et al., 1988). Modified nucleosides at position 37 are involved in correct structure formation of the anticodon loop. They improve the codon-anticodon interaction and the efficiency of tRNA (Björk, 1996).

Several modified nucleosides residing in different places on tRNA have an important function in reading frame maintenance. The presence of modified nucleosides reduces +1 frameshifting by either or both promoting efficient A-site selection and preventing peptidyl-tRNA slippage (Urbonavicius et al., 2001).

18 One such modification is N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine-37 (ms2io6A37) that influences the speed of some ternary complexes entering the A- site (Li et al., 1997). Formation of 2-thiocytidine-32 (s2C32) results in an altered anticodon loop structure (Baumann et al., 1985) by modulating a bifurcated hydrogen bond between cytidine at position 32 and nucleoside A38 (Auffinger and Westhof, 1999). Although a mutant lacking s2C32 exhibits a growth rate similar to the wild-type, the A-site selection rate for some of the tRNAs normally containing s2C32 is dependent on this thiolated nucleoside (Paper IV).

The modified nucleoside 4-thiouridine-8 (s4U8) can act as a sensor for UV- radiation as UV exposure induces the formation of a covalent bond between s4U8 and a C13 in some tRNAs (Favre et al., 1969; Thomas and Favre, 1975). This structural change results in poor aminoacylation of tRNAs and thereby triggers the stringent response (Ramabhadran and Jagger, 1976).

Iron-sulfur clusters and tRNA thiolation

Iron-sulfur cluster proteins Iron-sulfur clusters ([Fe-S] clusters) constitute one of the most ancient, ubiquitous, and functionally diverse classes of biological prosthetic groups (Beinert, 2000; Beinert et al., 1997; Kuchar and Hausinger, 2004). The underlying architectural element of [Fe-S] clusters is the [2Fe-2S] rhomb. Fe sites are generally tetrahedral and are most commonly coordinated by sulfur from inorganic sulfide and cysteine thiol groups (Rees and Howard, 2003), though sometimes histidine, aspartate, serine, or hydroxyle ligands are used (Gurrath and Friedrich, 2004). The simplest [Fe-S] clusters found in [Fe-S] proteins are [2Fe- 2S] and [4Fe-4S] clusters (Fig. 5). However, structures that are more complex exist, and besides Fe, other metals (Ni, Mo) may be contained.

CysC ys Cys Cys (a) (b) Cys Cys Cys Cys Cys Cys

Figure 5. The most common [2Fe–2S] (a) and [4Fe–4S] (b) clusters are coordinated to their protein partners by cysteine ligands (adapted from (Frazzon and Dean, 2003)). Iron and sulfur are shaded dark grey and light grey, respectively.

To date more than 100 different [Fe-S] protein classes have been described and have roles in mediating electron transport processes central in respiratory and photosynthetic electron transfer chains; redox processes of enzymes and proteins involved with carbon, oxygen, hydrogen, sulfur, and nitrogen metabolism; substrate binding and catalysis e.g. aconitase that converts citrate to isocitrate; environmental sensing and gene regulation, like in IRP (iron- responsive protein), FNR (fumarate-nitrate reduction protein), and SoxR (superoxide - response regulator) responding to changes in Fe, O2, and O2 , respectively; and, controlling protein structure (Beinert and Kiley, 1999; Frazzon and Dean, 2003; Kiley and Beinert, 2003). Radical SAM (S-adenosylmethionine) enzymes are a special class of several hundred [Fe-S] proteins. They have labile [4Fe-4S] clusters chelated by only three cysteines present in a conserved Cys-XXX-Cys- XX-Cys sequence, and require SAM for activity (Sofia et al., 2001). The combination of SAM, [Fe-S], and source of electrons is proposed to cleave SAM producing 5’-deoxyadenosyl radicals. The radicals then can initiate many metabolic reactions and biosynthetic pathways by hydrogen-atom abstraction (Cheek and Broderick, 2001; Fontecave et al., 2001, 2003, 2004; Jarrett, 2003; Marquet, 2001).

20 Synthesis of iron-sulfur clusters In vitro, [Fe-S] clusters spontaneously assemble in the correct conformation by incubation of apo-proteins with high concentrations of ferrous iron and sulfide under reducing conditions. However, free metal and sulfide ions are toxic in vivo. Thus, biologically [Fe-S] clusters are formed by protein directed activation and specific delivery of Fe and S to the assembly site (Frazzon and Dean, 2003). Pioneering studies by Dean and his colleagues of nitrogenase protein biosynthesis in Azotobacter vinelandii revealed that the inorganic S source for nitrogenase [Fe-S] clusters was the NifS protein, a pyridoxal phosphate- dependent L-cysteine desulfurase that creates enzyme bound persulfide (Zheng et al., 1993). Later it was shown, that A. vinelandii also possesses a second gene coding for a NifS-homologous protein. The gene was named iscS, and the operon it was located in an iron-sulfur-cluster operon (Zheng et al., 1998). Subsequently, similar operons comprising iscR-iscS-iscU-iscA-hscB-hscA-fdx (Fig. 6) were discovered in E. coli (Takahashi and Nakamura, 1999) and other bacteria, and homologues to those genes in eucaryotes (Lill and Kispal, 2000).

iscR iscS iscU iscA hscB hscA fdx

Figure 6. Organization of the E. coli isc-operon: iscR codes for the negative transcriptional regulator of the operon; iscS for pyridoxal-phosphate dependent cystein desulfurase; iscU for the [Fe-S] cluster assembly scaffold protein; iscA for an alternative [Fe-S] cluster assembly scaffold protein; hscB encodes a J type molecular co-chaperone; hscA a Hsp70-type molecular chaperone; and, fdx for [2Fe-2S] ferredoxin.

21 Lack of IscS dramatically decreases the specific activities of E. coli [Fe-S] proteins, verifying that IscS has a major role in [Fe-S] cluster formation (Schwartz et al., 2000; Tokumoto and Takahashi, 2001). Persulfide sulfur from IscS is transferred to IscU, which functions as a scaffold for [Fe-S] cluster assembly (Fig. 7) (Agar et al., 2000; Kato et al., 2002; Smith et al., 2001; Urbina et al., 2001). The precise nature of the interaction between IscS and IscU and the Fe source are unknown. Initial work proposed that IscA is an alternative scaffold to IscU for IscS-directed [Fe-S] cluster assembly (Krebs et al., 2001; Ollagnier- de-Choudens et al., 2001). However, structurally IscA and IscU are very different proteins (Adinolfi et al., 2004; Bertini et al., 2003; Bilder et al., 2004; Cupp- Vickery et al., 2004; Ding and Clark, 2004). The latest studies suggest that IscA can bind Fe under limited iron conditions and deliver it to IscU for [Fe-S] formation in vitro (Ding and Clark, 2004; Ding et al., 2004) but this remains to be investigated in vivo. The role of Fdx is unknown, but may involve an electron-

Direct sulfer transfer to other proteins not involved in [Fe-S] cluster formation

Chaperon functions as folding / unfolding Scaffold Cys Ala HscB Fe-S HscA IscU Fe-S Transfer of Fe-S proteins IscS Fe-S Fe -S Cysteine Fdx IscA desulfurase Redox Alternative scaffold function Fe -S or Fe donor IscR

Negative transcription

regulator

Figure 7. Network of proteins involved in biogenesis of [Fe-S] clusters.

22 transfer step required for [Fe-S] cluster synthesis. In vitro, IscA containing an [Fe-S] cluster forms a complex with Fdx and transfers its cluster to form the [2Fe-2S] holo-Fdx (Ollagnier-de-Choudens et al., 2001). Transfer of [Fe-S] clusters from the scaffold proteins to the target proteins is poorly understood, although proteins HscA and HscB are required (an Hsp70-type molecular chaperone and a J type molecular co-chaperone, respectively) (Hoff et al., 2000; Silberg et al., 2001). HscA specifically interacts with IscU by recognizing an amino acid sequence, stimulating its ATPase activity (Hoff et al., 2002). HscA- HscB chaperones are likely to be involved in either [Fe-S] cluster assembly on the IscU scaffold or in release of [Fe-S] clusters during maturation of target [Fe- S] proteins (Frazzon and Dean, 2003). The product of iscR gene located at the start of the isc operon (Fig. 6) regulates synthesis of Isc/Hsc proteins. IscR ligates an unstable [2Fe-2S] cluster required for isc operon promotor binding and repression of transcription (Schwartz et al., 2001). In such a way the [Fe-S] status of the cell is sensed and synthesis of [Fe-S] cluster formation machinery is adapted to the needs of the cell (Fig. 7). However, because IscS also provides sulfur for other S-containing biomolecules, isc-operon can not be completely turned-off. This issue requires more study.

Other proteins involved in iron-sulfur cluster metabolism E. coli contains two homologous proteins to IscS with related activities termed SufS (also known as CsdB) and CsdA (also known as CSD or YgdJ) (Kurihara et al., 2003; Takahashi and Tokumoto, 2002). These proteins might be responsible for the residual activity of [Fe-S] proteins in IscS lacking strains. SufS is contained within the sufABCDSE operon, regulated by iron and oxidative stress (Nachin et al., 2001; Patzer and Hantke, 1999; Zheng et al., 2001). The E. coli suf operon is specifically adapted to synthesize [Fe-S] clusters during iron or sulfur metabolism disruption by iron starvation or oxidative stress; i.e. under conditions where isc-encoded proteins cannot function effectively (Outten et al., 2004). SufC is an ATPase that binds with SufB and SufD to form the SufBCD complex (Nachin et al., 2003; Rangachari et al., 2002). SufS and SufE are shown

23 to form a new type of cysteine desulfurase, where sulfur from SufS is transferred to SufE (Loiseau et al., 2003; Ollagnier-de-Choudens et al., 2003a). This process is protected from the cellular environment and enhanced by the presence of the SufBCD complex (Outten et al., 2003) and SufA may function as a scaffold for [Fe-S] cluster assembly (Ollagnier-de Choudens et al., 2003b).

In S. enterica, in vivo [Fe-S] cluster metabolism of at least two [Fe-S] proteins requires the presence of ApbC (an ATPase) and ApbE (a periplasmic lipoprotein) (Skovran and Downs, 2003). Consistent with its sequence similarity to proteins that function in metal cofactor formation ApbC may facilitate Fe insertion into [Fe-S] clusters during their assembly or repair.

ISC machinery and tRNA thiolation A primary role for IscS is to provide sulfur for [Fe-S] clusters. However, IscS activity is also required for mobilization of sulfur necessary for biotin, thiamin, and Mo cofactor maturation, and tRNA thiolation (Fig. 7) (Fontecave et al., 2003; Frazzon and Dean, 2003).

Ten different thiolated nucleosides have been characterized so far (Auffinger and Westhof, 1998) and in various S. enterica tRNAs s2C, s4U, mnm5s2U, cmnm5s2U, and ms2io6A (Fig. 8) are present at positions 32, 8 or 9, 34, 34, and 37, respectively (Fig. 4). In E. coli, ms2i6A replaces ms2io6A (Fig. 8). Synthesis of thiolated nucleosides is a complex, multi-step process (especially for mnm5s2U/cmnm5s2U, and ms2io6A) (Fig. 9). For many years, knowledge about the tRNA thiolation was limited to that the sulfur originates from cysteine (Ajitkumar and Cherayil, 1988). Recently, IscS was demonstrated to be required for s4U formation (Kambampati and Lauhon, 1999; Lauhon and Kambampati, 2000). In further analysis of S. enterica and E. coli strains lacking IscS it was discovered that those mutants lack all thiolated nucleosides in their tRNAs (Lauhon, 2002; Nilsson et al., 2002).

24 s4U mnm5s2U ms2io6A

s2C cmnm5s2U ms2i6A

Figure 8. Structures of thiolated nucleosides in tRNA. Abbreviations: 2-thiocytidine (s2C), 4- thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5- carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), N-6-(4-hydroxyisopentenyl)-2- methylthioadenosine (ms2io6A), and N-6-isopentenyl-2-methylthioadenosine (ms2i6A).

During s4U synthesis, sulfur is transferred from cysteine to IscS forming a persulfide at Cys328 in the IscS active site. The persulfide sulfur from IscS is transferred to a cysteine in ThiI, which in turn transfers the sulfur to a uridine in tRNA position 8 (Fig. 9) (Kambampati and Lauhon, 2000; Mueller et al., 2001; Palenchar et al., 2000; Wright et al., 2002). Alternatively, the sulfur from ThiI may be transferred to ThiS, which introduces the sulfur to the thiazole part during thiamine formation (Taylor et al., 1998; Webb et al., 1997). Thus, the syntheses of thiamine and s4U are metabolically linked.

Analysis of mnm5s2U/cmnm5s2U synthesis in the tRNA wobble position revealed that the thiolation step is independent of the presence of the mnm5- or cmnm5- moiety ((c)mnm5- designates the combination of the two moieties). The first step in the formation of the mnm5-side chain is the formation of cmnm5-group by MnmE and GidA. MnmC catalyzes the formation of the mnm5-side chain in a two-step re-arrangement of cmnm5 (Fig. 9) (Hagervall et al., 1987). Initial studies

IscS ThiI U8 s4U8

IscS TusA+TusBCD+ MnmE + GidA MnmC MnmA U34 cmnm5U mnm5U mnm5s2U34

TtcA C32 s2C32

IPP IscS MiaA MiaB MiaE A37 i6A ms2i6A ms2io6A37

Figure 9. Biosynthetic pathways for the formation of thiolated nucleosides.

of the thiolation step involved in (c)mnm5s2U formation indicated that the persulfide sulfur of IscS (as in ThiI) may also be transferred to the acceptor protein MnmA, which in turn transfers the sulfur to a uridine in the tRNA (Kambampati and Lauhon, 2003). However, the transfer of sulfur from IscS to MnmA (the last protein in the biosynthetic pathway and meeting the tRNA) may be via TusA (T. Suzuki, personal communication). The protein complex TusBCD is also required for the s2U formation as it enhances the TusA activity (Fig. 9).

In a subset of tRNAs that read codons starting with U nucleoside A37 receives isopentenyl group at position 6 from isopentenyl pyrophosphate (IPP) in the reaction catalysed by MiaA (Leung et al., 1997; Moore and Poulter, 1997; Rosenbaum and Gefter, 1972). The product of the miaB gene participates in the later methylthiolation of i6A (Esberg and Björk, 1995; Esberg et al., 1999). MiaB contains an oxygen-labile [Fe-S] cluster and is a member of the Radical SAM

26 protein superfamily (Pierrel et al., 2002, 2003; Sofia et al., 2001). Enzymes in this superfamily form C-S bonds via sulfur insertion by radical mechanisms (Fontecave et al., 2003) although the sulfur source for the thiolation is unknown (possibly persulfide sulfide or from the [Fe-S] cluster). It is also unknown whether MiaB is only responsible for the thiolation step, or both the thiolation and methylation of i6A. In S. enterica the MiaE protein is required for the subsequent hydroxylation of the i6-group of ms2i6A (Fig. 9) (Persson and Björk, 1993).

Until recently, the mechanism for s2C32 synthesis was unknown. In this thesis, I present data that the product encoded by the ttcA gene is required for s2C synthesis (Fig. 9) (Paper IV).

In conclusion, the thiolation of U in positions 2 (for (c)mnm5s2U) and 4 (for s4U) involves multiple persulfide sulfur transfers, whereas the methylthiolation reaction in the synthesis of ms2io6A is different. Interestingly, studies of an iscS deletion mutant revealed that the synthesis of s2C and ms2io6A but not (c)mnm5s2U and s4U could still occur at lower rates, suggesting a residual level of the mobilization of sulfur via an IscS independent pathway (Lauhon, 2002; Nilsson et al., 2002).

27 AIMS OF THIS WORK

• To identify and characterize the frameshift suppressor sufF, and determine how it induces the frameshifting.

• To isolate and characterize new suppressor inducing frameshifting in the hisC mutated gene (hisC3072).

• To identify the link between the isc-operon encoded proteins and the appearance of thiolated nucleosides in tRNA.

• To identify the gene responsible for cysteine thiolation in tRNA, investigate the protein it codes, and determine the cellular function of s2C32.

28 RESULTS AND DISCUSSION

The role of different tRNAs, identification of enzymes involved in tRNA modification, and performance of tRNA in different translational steps has been extensively investigated in our group. A lot of data has been obtained by analysing the nature of frameshifting suppressors in the model organism S. entrica. My work was not an exception. The operon most frequently used in our studies codes for histidine biosynthetic genes. A collection of frameshift mutations in genes hisA, hisB, hisC, hisD, and hisF was created many years ago (Riddle and Roth, 1970). These mutants are dependent on added histidine for growth, though in the presence of frameshift suppressors they become His+. Using this His-/His+ system I have characterized several mutations altering different constituents of the cell. All the mutants directly or indirectly affect the translational machinery (Fig. 10).

Frameshift mutation in a his gene (His- phenotype)

Selection for the phenotype change

from His- to His+

Extragenic suppressor mutations (His+ phenotype)

Arg tRNA 5 mnm UCU L9 IscU TtcA

Figure 10. Strategy of mutant selection. Isolation and characterization of each mutant will be discussed in the text.

29 Frameshifting induced by decreased stability and reduced aminoacylation of a minor arginine tRNA (Paper I)

Arg The sufF mutant has an altered tRNA mnm5UCU The frameshift suppressor sufF was isolated in S. enterica by Dr. J.Roth’s laboratory (Riddle and Roth, 1970), mapped, and shown to be recessive to the wild type gene (Riddle and Roth, 1972a). sufF and sufD mutations suppressed the same frameshift site in the hisC gene (hisC3072) and sufD affects the structure of a glycine-tRNA (Riddle and Roth, 1972b). Based on these facts it was proposed that sufF probably codes for an enzyme involved in glycine-tRNA modification. The evidence that glycine-tRNA from the sufF mutant had altered chromatographic properties strengthened this suggestion (Riddle and Roth, 1972b). With this knowledge, I started my studies of sufF. The first task was to identify sufF gene. With the help of mutations in purE and fimC genes (both transductionaly linked to sufF), I could narrow down the location of sufF on the chromosome. From the S. enterica gene bank, a complementing plasmid was selected that changed the phenotype of a double hisC3072 sufF mutant from His+ to His-. Among the other genes present on the plasmid was the argU gene encoding a minor arginine tRNA. Sequencing of argU in the sufF mutant

Arg tRNA 5 revealed that the frameshift suppressor mutation altered mnm UCU . This result was in sharp contrast to our initial presumptions.

New suppressors were isolated using localized mutagenesis bearing other

Arg tRNA 5 alterations in mnm UCU . The changes inducing a +1 frameshifting were in the TΨC stem (C61U, G53A) and loop (C56U), as well as in the anticodon stem (G39A) and loop (s2C32U) (Paper I, Fig. 2). Residues C61 and G53 are highly conserved in all tRNA species and are involved in stabilization of the characteristic TΨC loop structure (Dirheimer et al., 1995). Mutations C61U and G53A probably compromised that structure. Base C56 is also highly conserved and participates in the tertiary base pair G19-C56 that locks the D and TΨC loops

30 together (Dirheimer et al., 1995). The C56U mutation, which disrupted bonding to G19 could in turn alter the interference with the adjacent nucleotide D20 (discussed later). The G39A mutation disrupted the first base pair of the anticodon stem that closes the anticodon loop, altering a 7-nucleotide-long anticodon loop into a 9-nucleotide-long anticodon loop. The s2C32U mutation caused both a lack of thio-modification and a different base in the anticodon loop. Additional experiments revealed that lack of the thio-modification alone could not be responsible for the suppressor phenotype. Thus, the effect induced by change of C32 to U32 may be by modification of the non-canonical bifurcated hydrogen bond between residues 32 and 38 that shapes the anticodon loop (Auffinger and Westhof, 1999). Also, one new mutation was identified in the promoter region of argU gene that altered the -10-consensus sequence (from TATAAT to TACAAT) important for transcription initiation.

Arg Arg tRNA 5 tRNA 5 mnm UCU and/or arginyl- mnm UCU levels are decreased in the mutants As the isolated mutations changed the secondary and/or tertiary structure of the

Arg tRNA they could render tRNA mnm5UCU less stabile and increase its degradation.

Arg tRNA 5 Therefore, I monitored the levels of mnm UCU in different mutant strains by Northern blot hybridization. The C61U, G53A, and C32U mutations decreased

Arg tRNA 5 the amount of mnm UCU in the cells 2- to 3-fold as compared to the wild-type, but no change was induced by the C56U and G39A mutations. The T-10C

Arg promoter mutant had a three-fold reduced expression of tRNA mnm5UCU , consistent with the observed reduction of transcription in other TATAAT to TACAAT -10- consensus sequence mutants (Gaal et al., 1989; Youderian et al., 1982).

C35, U/G36, A/G73, and D20 in tRNAArg are the major positive recognition elements for aminoacylation by ArgRS (Giegé et al., 1998). The comparison of

Arg tRNA 5 aminoacylated versus non-acylated mnm UCU amounts in all the mutants

Arg revealed that the arginylation levels of tRNAmnm5UCU decreased in the C56U,

31 C61U, G53A, and G39A mutants. The effect of the G39A mutation on the overall anticodon loop structure, and the effects of C61U and G53A mutations on

Arg tRNA 5 TΨC loop structure impaired the binding of mnm UCU to ArgRS. Mutation C56U, which induced the strongest effect (more than 2-fold decreased

Arg aminoacylation) in addition to changing the tertiary structure of tRNA mnm5UCU could alter the D20 identity element involved in direct chemical interaction with ArgRS.

Glu tRNA 5 2 Peptidyl- mnm s UUC frameshifts when the ribosome is pausing Sequencing revealed that the frameshift mutation in gene hisC3072 is an insertion of an extra G in a run of Gs, creating the sequence GGG GAA AGA

(N)87 UGA (the zero reading frame formed following the insertion and the inserted G (underlined) is presented). The mutation resulted in the synthesis of a

Arg truncated HisC protein. tRNAmnm5UCU recognizes the rare codon AGA. As

Arg Arg tRNAmnm5UCU is a minor tRNA, the cellular quantities of arginyl- tRNAmnm5UCU are low, in argU - suppressors the quantities were decreased even further. How could this influence the translation? To answer that the peptide sequence encoded by the suppressed frameshift site was examined. The sequence was Gly(GGG)- Glu(GAA)-Asp(GAC) over the GGG-GAA-AGA-CNN mRNA sequence

Glu suggesting that the peptidyl- tRNA mnm5s2UUC frameshifts when the ribosome contains the AGA codon in the A-site. Moving one nucleotide rightwards brings a GAC codon (decoded by tRNAAsp) into the A-site. Therefore, in the suppressor

Arg strains the levels of charged tRNAmnm5UCU are low and this induces the ribosome

Glu to pause, which in turn allows the peptidyl- tRNAmnm5s2UUC to shift into +1 reading frame (Paper I, Fig.1). These results were in agreement with the previous observation that codons deprived for the aminoacylated tRNAs cause frameshifting (Lindsley and Gallant, 1993; Weiss et al., 1988).

32 Ribosome slippage induced by defects in the ribosomal protein L9 (Paper II)

Frameshift mutation is suppressed by mutations in ribosomal protein L9 The same frameshift mutation in the hisC gene (hisC3072) containing an extra G in a run of Gs was used in another selection for external frameshift suppressors expected to generate mutations affecting the synthesis of a modified nucleoside e.g. cmnm5U. His- cells carrying hicC3072 were infected by phages mutagenized with hydroxylamine and His+ clones selected. One such His+ clone (strain GT5167) was saved for further analysis. The suppressor mutation in GT5167 was weakly linked to the vacB2530::Tn10dTc insertion. Transductional mapping of the suppressor to other chromosomal markers located in the same chromosomal area positioned the mutation in the ribosomal operon containing rpsF, rpsR, and rplI genes that code for ribosomal proteins S6, S18, and L9, respectively. The gene sequences revealed that the suppressor mutation was in the rplI gene and caused the amino acid substitution G24D in the large ribosomal subunit protein L9. Protein L9 has a highly elongated shape consisting of two globular domains (N- and C-domain) separated by a connecting helix (Hoffman et al., 1996). In the ribosome, it protrudes perpendicularly from the surface of the large subunit near the mRNA exit channel (Yusupov et al., 2001). Local mutagenesis was employed to test if other L9 alterations could induce similar frameshift suppression. The mutator activity of DinB (DNA polymerase IV) created nine new L9 mutants that suppressed the His- phenotype of the hisC3072 mutant. The L9 mutants contained amino acid changes in the N-domain (G24D) or C-domain (I93S, A101D, G125V, and F131S) mostly affecting the conserved structural elements of the protein. In addition, I isolated frameshift mutations in rplI altering either the final portion of the C-terminus, or removing the C-terminal domain with or without the L9 connecting helix. L9 deletion mutant was created by deleting the entire L9 gene and also shown to have suppressor phenotype. The absence of L9 and analyzed base substitution and truncation mutants of L9 induced a similar

33 level of suppression. Which of the mutated forms of L9 are incorporated into the ribosome needs to be investigated.

The mechanism of ribosomal slippage induced by L9 mutants is unknown All L9 mutants isolated in this study were selected to synthesize a functional HisC protein. Formation of a functional HisC was possible if the ribosomes slipped to the +1 frame during translation of the hisC3072 mRNA. It could occur by +1 frameshifting to the overlapping codon or by bypassing to codons located further downstream in the +1 reading frame (if the protein synthesized by such bypassing was active). To gain an insight into the mechanism behind suppression of frameshift mutation a sequence of the peptide synthesized from an mRNA having a similar frameshift site as that present in hisC3072 was determined in a L9 lacking strain. So far, two trials resulted in two different mixed amino acid sequences, neither of which resembled the sequence coded by the test mRNA. This implies that L9 deficient ribosomes slip to the +1 frame at multiple sites and land at various landing sites, producing different peptides. The suggestion for different slippage events is consistent with results obtained in studies of the effect L9 mutants have on programmed ribosomal hopping in the translation of bacteriophage T4 gene 60 mRNA. It was demonstrated that L9 defects increase +1, -1 frameshifting, and stop-hopping with diverse landing sites. It was proposed that L9 influences the direction of mRNA movement through the ribosome (Herr et al., 2000, 2001, 2004).

One could argue that mutations in L9 render the ribosome prone to slip without any specific stimulus. However, such mutants would be expected to have at least a slightly impaired growth rate, which was not the case for the colonies of L9 mutants. Besides, some specificity was observed by comparing the suppression of different frameshift mutations (all His-) containing either a run of Cs or a run of Gs in various contexts. L9 mutant G24D suppressed only three frameshift mutations from eight tested. Common signals inducing the L9 deficient ribosome slippage could not be sought, as not all of the tested frameshift mutations are

34 sequenced. Also, methods for monitoring the diversity of ribosome dissociation and landing sites in the hisC3072 mRNA have to be improved.

Enzymes required for tRNA thiolation (Papers III and IV)

A former member of our group, Qiang Qian, was also using the His-/His+ system

Pro to search for mutants in proL (codes for tRNAGGG ) that induces suppression of a frameshift mutation in the hisD gene (hisD3749) which had en extra C in a run of Cs. The results suggested that at the frameshifting site CCC-UGA present in

Pro Pro tRNA 5 hisD3749 a defective GGG allowed near-cognate tRNA cmo UGG to decode CCC and shift to the +1 frame in the P-site, thereby inducing a His+ phenotype (Qian et al., 1998). When Qiang mutagenized the chromosome in the proL region and selected His+ clones in the presence of the hisD3749 mutation, one clone was different from all the others. This clone grew slowly and had decreased amounts of the thiolated nucleosides s2C and ms2io6A in the tRNA. However, these

Pro thiolated modifications are not normally present in tRNA GGG . None of the new phenotypes was linked to proL and the His+ phenotype. My task was to identify the gene responsible for these peculiarities. Why was the synthesis of two modified nucleosides affected? If there was a blockage early in the sulfur transfer, why didn’t it affect all five thiolated nucleosides that were present in the cell?

Formation of s2C and ms2io6A in tRNA requires functional iron-sulfur proteins (Paper III) Transductional mapping placed the mutation inducing slow-growth within the iscR-iscS-iscU-iscA-hscB-hscA-fdx operon coding for ISC machinery proteins. This machinery assembles and delivers [Fe-S] clusters to target [Fe-S] apo- proteins. One member of the operon was already connected to the process of tRNA thiolation. IscS is required for the synthesis of all five thiolated nucleosides by mobilizing and distributing sulfur to enzymes that catalyze the

35 sulfur insertion steps (Lauhon, 2002; Nilsson et al., 2002). After sequencing the entire isc operon in the slow growing mutant with decreased concentrations of s2C and ms2io6A in the tRNA, only a single point mutation in the iscU gene was identified. This mutation caused an S107T amino acid change in the IscU protein. IscU serves as a scaffold for the transient [Fe-S] cluster ligated by three conserved cysteines in positions 37, 63, and 106 (Zheng et al., 1998) and interacts with the chaperone HscA that recognizes a specific amino acid region LPPVK (residues 99 to 103) present in IscU (Hoff et al., 2002). As the S107T point mutation is only 3 amino acids away from the recognition site it could impair the recognition of IscU by HscA. The change S107T could also interfere with [Fe-S] cluster coordination by adjacent C106. Irrespective of which or both of these alternatives was valid, the point mutation elicited a powerful effect on IscU function as it induced a similar phenotype as the complete absence of IscU protein in the ∆iscU mutant strain. Both the point mutation in IscU or lack of IscU decreased the tRNA level of ms2io6A more then 10-fold and the level of s2C more then 2-fold. The levels of (c)mnm5s2U and s4U in the tRNA remained unchanged. hscA and fdx deletion mutants gave analogous results. The strains containing iscU52(S107T), ∆iscU, ∆hscA, or ∆fdx mutations all grew slowly. Mutations in the same proteins (IscU, HscA, and Fdx) have severely decreased [Fe-S] enzyme activities (Tokumoto and Takahashi, 2001). Therefore, I concluded that active [Fe-S] proteins are required for formation of s2C and ms2io6A, which appears to be in contrast for synthesis of (c)mnm5s2U and s4U. In the latter case, the persulfide formed in IscS is successively transferred to accepting proteins until the sulfur is inserted in uridines of tRNA.

A candidate [Fe-S] enzyme for ms2io6A synthesis has been recently described. MiaB is involved in the modification of A37 and requires the [Fe-S] cluster for activity (Pierrel et al., 2002). A mutation in any of the cysteines from the [Fe-S] cluster coordinating Cys-XXX-Cys-XX-Cys sequence halts production of ms2io6A in tRNA. However, nothing was known regarding the enzymes involved in the synthesis of s2C.

36 TtcA is involved in thiolation of cytidine at position 32 and may hold an iron-sulfur cluster (Paper IV) Since an E. coli strain lacking s2C (but containing ms2i6A) has already been described (Qian et al., 1998), a search for the gene causing this deficiency was initiated. Hfr mapping and subcloning from the complementing lambda phage DNA revealed that the ydaO gene is required for s2C synthesis. This gene was renamed ttcA, for “two-thio-cytidine”. As all the other tRNA thiolation work was performed in S. enterica we mutated a ttcA homologue in this bacterium. tRNA hydrolysates from strain ∆ttcA1 lacked s2C, therefore we concluded that TtcA was also involved in the synthesis of s2C in S. enterica. Compared with the wild type, the strain lacking s2C had no growth rate or general and sulfur metabolism differences. Closer examination of the amino acid sequences in TtcA homologues revealed that they contained several conserved motifs. The SGGKDS motif present in TtcA is characteristic of ATPases in the PP-loop superfamily, and the Cys-XX-Cys motif is characteristic of proteins in the thioredoxin superfamily. Since cysteines are important for MiaB function by ligating the [Fe-S] cluster, their role in the TtcA Cys-XX-Cys motif was investigated. Changing both cysteines to alanine and each of them separately to serine proved that both the cysteines in the TtcA motif were essential for s2C formation. TtcA from S. enterica contains one additional semi-conserved Cys- XX-Cys motif in the C terminus and three other cysteines within the peptide. Biochemical characterization would be required to find if TtcA contains a [Fe-S] cluster, but it has enough cysteines to coordinate such a cluster. Data regarding ISC machinery mutants (Paper III) suggests that some of the protein(s) in s2C synthetic pathway requires [Fe-S] cluster for its function.

Lack of IscA specifically decreases the levels of ms2io6A but not that of s2C (Paper III) Analysis of isc operon gene deletions revealed that a deletion of iscA differed from the other mutants. Analysis of total tRNA purified from that strain revealed that the presence of IscA was only critical for ms2io6A synthesis, since its level

37 was decreased 2-fold, whilst the levels of s2C, (c)mnm5s2U, and s4U were unchanged compared to the wild-type. Since iscA is affecting the assembly of [Fe-S] clusters (although the effect was much less dramatic than that of the iscU, hscA, or fdx genes) it was concluded that IscA protein appears to improve the efficiency of [Fe-S] cluster assembly (Tokumoto and Takahashi, 2001). Our results also suggest that IscA could have a role in the repair of [Fe-S] clusters. MiaB has an oxygen-labile cluster, which is a common feature for proteins belonging to the Radical SAM family. Upon oxidation the labile [4Fe-4S] clusters lose iron and generate [3Fe-4S] and [2Fe-2S] forms, resulting in inactive forms of the enzymes (Cheek and Broderick, 2001). This transition in cluster forms has also been demonstrated in MiaB (Pierrel et al., 2002, 2003). Therefore, in aerobic environments, oxygen-labile clusters require more efficient repair and could explain why the absence of IscA only affected the synthesis of ms2io6A but not that of s2C, provided that the protein(s) in the s2C pathway have a more stabile [Fe-S] cluster. The different functions suggested for IscA including acting as an alternative scaffold for transient [Fe-S] cluster assembly or binding and delivering iron (more relevant here) could facilitate the repair of MiaB. Possibly, growth under anaerobic conditions or in iron-enriched medium could suppress the effect induced by lack of IscA. Such suppression was demonstrated for other oxygen-sensitive [Fe-S] enzymes (Benov and Fridovich, 1998; Skovran and Downs, 2003).

Two pathways for sulfur in tRNA thiolation (PaperIII) Based on the results, two principally distinct pathways for biosynthesis of thiolated nucleosides can be postulated (Fig. 11). The initial step common for all five thio-modifications is IscS mediated mobilization of sulfur from cysteine by forming a persulfide bound to the enzyme. Then the pathways diverge. During s4U and (c)mnm5s2U syntheses sulfur is directly transferred from IscS to the tRNA modifying enzyme ThiI or intermediate proteins successively transferring sulfur to tRNA modifying enzyme MnmA, respectively, without the participation of [Fe-S] proteins. The second pathway leads to the syntheses of s2C and ms2io6A

38 and besides IscS, other constituents of the ISC machinery are needed, as it includes proteins containing [Fe-S] clusters. Such [Fe-S] protein is MiaB required for ms2io6A formation. We suggest that TtcA could be the [Fe-S] protein required for s2C formation or alternatively, an unknown [Fe-S] protein(s) could be involved in s2C formation before or after the action of TtcA.

In support for the existence of two separate pathways is the recent observation that synthesis of s4U and (c)mnm5s2U is completely dependent on the IscS as sulfur donor, whereas an inefficient synthesis of s2C and ms2io6A occurs in the absence of IscS (Lauhon, 2002; Nilsson et al., 2002). This may be due to a residual level of [Fe-S] cluster biosynthesis in iscS deficient strains.

ThiI s4U

5 2 MnmA (c)mnm s U

Cysteine IscS TtcA s2C

MiaB ms2io6A

[Fe-S] IscA

IscU HscA Fdx

Figure 11. Model for sulfur trafficking in tRNA thiolation. Solid lines represent experimentally verified pathways, dashed lines indicate hypothetical interactions between proteins. Wide arrows indicate the assembly and delivery, or repair of [Fe-S] clusters.

39 Consistent with the proposed model, specific IscS mutants were recently isolated. Two residues in the flexible loop containing the active site cysteine C328 selectively affected in vivo [Fe-S] cluster biosynthesis without detectably affecting persulfide delivery destined for s4U and mnm5s2U formation (Lauhon et al., 2004). In vitro studies suggest that sulfur incorporation in [Fe-S] clusters also proceeds via a persulfide transfer between IscS and IscU (Kato et al., 2002; Smith et al., 2001; Urbina et al., 2001). The finding of specific mutants altering the transfer of the same persulfide to IscU but not ThiI implies that during in vivo [Fe-S] cluster biosynthesis the function of IscS may be more complex than just a persulfide transfer to IscU, and confirms the dual role of IscS in thionucleoside biosynthesis.

The origin of the sulfur inserted in tRNA for s2C and ms2io6A formation is unknown. As enzymes catalyzing sulfur insertion for both modifications require [Fe-S] clusters for activity, the cluster itself could provide the sulfur. Alternatively, the sulfur may be from persulfide formed by a hypothetical enzyme analogous to IscS. This question has been intensively studied by several groups investigating BioB, a Radical SAM enzyme similar to MiaB that participates in sulfur introduction during synthesis of biotin. On the one hand BioB itself may have a cysteine desulfurase activity forming persulfide that could be used in sulfur insertion reaction (Ollagnier-De-Choudens et al., 2002). Based on those facts the mechanism of ms2io6A synthesis was inferred to involve both persulfides and radical formation by [Fe-S] cluster and SAM (Fontecave et al., 2003). On the other hand, another group of investigators argues that BioB lacks a desulfurase activity and it is its second [Fe-S] cluster that provides the sulfur possibly by degradation of the [Fe-S] cluster and the protein-bound polysulfide or persulfide formation (Cosper et al., 2004; Jameson et al., 2004). However, only one [4Fe-4S] cluster has been detected in MiaB (Pierrel et al., 2003). Future experiments are required to solve this enigma and suggest how results obtained from BioB could be inferred to explain the formation of ms2io6A by MiaB.

40 tRNA thiolations and frameshifting Several tRNA modifications have the common function of improving reading frame maintenance (Urbonavicius et al., 2001) by promoting efficient A-site selection and/or preventing peptidyl-tRNA slippage in a tRNA specific manner and depending on the codon read. s4U8 is the most prevalent thionucleoside in tRNA. However, there is no data regarding the influence of s4U on reading frame maintenance. s2C is present in position 32 of only four E. coli and S. enterica tRNA species

Arg Arg Arg Ser tRNA tRNA tRNA 5 tRNA ( ICG , CCG , mnm UCU , and GCU ) (Sprinzl and Vassilenko, 2003). Presence of s2C32 moderately increased the selection rate of aminoacyl- tRNA at the ribosomal A-site at the AGG codon but not at AGA codon, or at any of the CGN cognate codons. s2C32 present in peptidyl-tRNA interfered with aa-

Arg 2 tRNA selection at the A-site codon. Surprisingly, tRNA ICG containing s C32 had a decreased rate of CGA translation, revealing that this modified nucleoside may

Arg negatively modulate the activity of tRNA ICG (Paper IV).

(c)mnm5s2U is present in the wobble position in tRNAs specific for Gln, Lys, and Glu. Lack of the s2-group decreased A-site selection and increased P-site slippage for tRNALys and tRNAGln, though for tRNAGln the effect was codon specific (Urbonavicius et al., 2001). ms2io6A is present in eight tRNAs reading codons starting with U, except

Ser 2 tRNA GGA . Presence of ms -group had little or no effect for A-site selection in a tRNA-dependent manner, whereas it strongly prevented P-site slippage for tested tRNAs (Li et al., 1997; Urbonavicius et al., 2001).

The knowledge of various effects elicited by lack of individual thio- modifications in tRNA can be utilized. In hisC3737 a potential +1 frameshifting

41 site (CCC-CAA-UAA) exists rendering this strain His-. This site may be

Pro suppressed by near-cognate peptidyl- tRNA cmo5UGG , which decodes CCC and shifts

Glu to +1 frame when the ribosome pauses due to slow entry of the tRNAmnm5s2UUC to

Gln the CAA codon in the A-site. Lack of a thio-modification in tRNAmnm5s2UUC impairs the A-site entry and induces +1 frameshifting resulting in a His+ phenotype. This system enabled the isolation of an iscS mutant that had decreased levels of (c)mnm5s2U, and also affected the synthesis of the rest of tRNA thio-modifications (Nilsson et al., 2002). Designing specific sites where the required frameshifting would be dependent on the absence of either (c)mnm5s2U or ms2io6A the isolation of mutants lacking those modifications becomes possible. According to the proposed sulfur trafficking model (Fig. 11), lack of (c)mnm5s2U could be induced by mutations in iscS and also influence other thiolations, or by mutations altering the (c)mnm5s2U pathway specifically. In contrast, lack of ms2io6A could be induced by mutations in any of the genes in the isc operon also influencing the presence of s2C, or by mutations in the genes altering the pathway specific for ms2io6A. The occurrence of IscS substitutions specifically impairing [Fe-S] clusters assembly (Lauhon et al., 2004) implies that different residues in IscS could be responsible for the transfer of persulfide to other partners than IscU. From the point of tRNA thiolations such mutations would effect s4U and (c)mnm5s2U but not ms2io6A and s2C. The search for this kind of mutants is an ongoing project in our laboratory.

42 CONCLUSIONS

Arg tRNA 5 • The +1 frameshift suppressor sufF is a mutation in the minor mnm UCU

Arg gene argU. Mutants in tRNA mnm5UCU read A-site AGA codon inefficiently due

Glu to decreased stability and reduced aminoacylation, allowing the tRNAmnm5s2UUC residing in ribosomal P-site to slip forward on the mRNA.

• Single base substitutions, truncations, and deletion of ribosomal protein L9 induces ribosome slippage to the +1 frame by an unknown mechanism.

• Formation of thiolated nucleosides in tRNA occurs in two principally distinct pathways, an [Fe-S] cluster-dependent pathway for synthesis of ms2io6A and s2C and [Fe-S] cluster-independent pathway for synthesis of s4U and (c)mnm5s2U.

• TtcA protein is involved in the formation of s2C32 in tRNA. Cysteines in the TtcA conserved Cys-XX-Cys motif are indispensable for synthesis of this modification.

43 ACKNOWLEDGEMENTS

First of all I would like to express my gratitude to my supervisor, Glenn Björk, for bringing me into the exciting world of science. His sparkling enthusiasm and never-ending ideas guided me both through less successful and more joyful projects of my Ph.D.

Thanks to present and past members of group GB for sharing my years here in the lab that started in Ji-nong’s, Qiang’s, Ólafur’s, Birgitta’s, Jaunius’s, and Jerome’s companionship eventually exchanged by Hans, Peng, Jocke and Mike. Especially thanks to Jaunius, Hans and Peng for sharing the last-year-student worries. Kerstin, Kristina and Gunilla deserve a great credit for being the corner stones of our group. Thanks also for your helping hands when my time was running out.

I would like to acknowledge Tord Hagervall, Mikael Wikström, Olof Persson, Anders Bystöm, Kerstin Stråby and members of their groups for scientific discussions, Mark Dopson for editing this thesis.

Secretaries, dishwashing, media and P-A with Johnny are acknowledged for their valuable everyday services.

I am extremely grateful to all Lithuanian friends for being my family here in Umeå, for sharing all my joys and sorrows.

I would like to thank my parents for all the care and support I got, my sisters for being curious for how is it going for me. Being far away from Lithuania I always felt you are with me.

Arunas, Gabija and Joris are part of me and I am part of them. I am so grateful to fortune that they exist.

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