Studies on Translation Initiation and Termination in Escherichia Coli

Studies on translation initiation and termination in

Escherichia coli

Georgina Ibrahim Isak

Department of Genetics, Microbiology and Toxicology

Stockholm University, Sweden 2012

Doctoral thesis 2012 Department of Genetics, Microbiology and Toxicology Stockholm University SE-106 91, Sweden

©Georgina Ibrahim Isak, Stockholm 2012 ISBN 978-91-7447-388-9

Printed in Sweden by Universitetsservice US-AB, Stockholm 2012 Distributor: Department of Genetics, Microbiology and Toxicology

Cover illustration: Structure of the ribosome. The 70S ribosome with mRNA and A- P- and E- site tRNAs. Picture adapted with permission from [1]. Copyright © 2009, Rights Managed by Nature Publishing Group.

Till min familj

4 Abstract

Translation initiation factor 1 (IF1) has been shown to be an RNA chaperone. In order to find functional interactions that IF1 may have with rRNA, we have isolated second‐site suppressors of a cold‐sensitive IF1 mutant. Joining of the ribosomal subunit seems to be affected in the IF1 mutant strain and the suppressive effect is a consequence of decreasing the available pool of mature 50S subunits. The results serve as additional evidence that IF1 is an RNA chaperone and that final maturation of the ribosome takes place during translation initiation.

In this study we have also investigated the effect of a cold‐sensitive mutant IF1 or kasugamycin addition on gene expression using a 2D gel electrophoresis technique. The effect is much more dramatic when cells are treated with kasugamycin compared to mutant IF1. The ybgF gene is uniquely sensitive to the IF1 mutation as well as the addition of kasugamycin. This effect on the native gene could be connected with some property of the TIR sequence of ybgF and supports the notion that kasugamycin addition and the IF1 cold‐sensitive mutation have a similar TIR‐specific effect on mRNA translation.

Finally we have isolated a suppressor of a temperature‐sensitive mutation in ribosomal release factor 1 (RF1) to shed more light on the translation termination process. The suppressor mutation is linked to an IS10 insertion into the cysB gene and results in a Cys‐ phenotype. Our results suggest that suppression of the thermosensitive growth is a consequence of the mnm5s2U hypomodification of certain tRNA species. The ability of mnm5s2U hypomodified tRNA to induce frameshifting may be responsible for the suppression mechanism and it supports the hypothesis that modified nucleosides in the anticodon of tRNA act in part to prevent frameshifting by the ribosome.

List of publications

The thesis is based on the following publications

I. Jaroslav Belotserkovsky, Georgina Isak, Leif A. Isaksson (2011) Suppression of a cold‐sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region. FEBS J., 278(10):1745‐56

II. Sergey Surkov, Georgina Isak, Leif A. Isaksson Influences of a mutated translation initiation factor IF1 or kasugamycin on Escherichia coli gene expression. (Submitted)

III. Georgina Isak, Monica Rydén‐Aulin (2009) Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature‐sensitive phenotype caused by mutant release factor 1. J. Bacteriol.,191(5):1604‐9

Permissions to reproduce papers I and III were kindly obtained from the publishers

Table of contents

1. Introduction…………………………………………………………………………... 9 1.1 Bacterial translation process……………………………………………………….. 9

1.2 The Bacterial ribosome and its subunits………………………………………….. 10

1.2.1 The small ribosomal subunit………………………………………………...... 11

1.2.2 The large ribosomal subunit…………………………………………………… 11

1.3 Assembly of ribosomal subunits…………………………………………………... 13

1.3.1 rRNA maturation and modifications…………………………………………. 13

1.3.2 Binding of ribosomal proteins and rRNA folding…………………………... 15

1.3.3 Ribosomal assembly factors………………………………………………...... 18

DEAD‐ box proteins, GTPases, Chaperones and maturation factor……...... 19

1.4 Bacterial translation initiation…………………………………………………...... 21

1.4.1 The translation initiation region in messenger RNA………………………... 22

1.4.2 The initiator transfer RNA (itRNA)…………………………………………… 22

Transfer RNA modification……………………………...... 23

1.4.3 Initiation factor 1 (IF1)………………………………………………………….. 24

1.4.4 Initiation factor 2 (IF2)………………………………………………………….. 26

1.4.5 Initiation factor 3 (IF3)………………………………………………………….. 27

1.4.6 The antibiotic kasugamycin as a translation initiation inhibitor…………… 28

1.5 Bacterial translation termination and recycling………………………………….. 30

1.5.1 Class 1 release factors…………………………………………………………. . 30

2. Results and discussion……………………………………………………………… 34

2.1 Paper I……………………………………………………………………………….. 34

2.2 Paper II……………………………………………...... 36

2.3 Paper III…...... 37

3. Concluding remarks………………………………………………………………… 39

4. Acknowledgements…………………………………………………………………. 40

5. References…………………………………………………………………………...... 41

Abbreviations

ASD anti Shine‐Dalgarno sequence A‐site aminoacyl‐tRNA binding site ASL anticodon stem‐loop CP central proturberance DASL dimethyl‐A stem‐loop DR The downstream region E‐site exit‐tRNA binding site EF‐Tu Elongation factor Tu fMet ‐tRNA fMet formylated initiator tRNA GDP guanosine diphosphate GTP guanosine triphosphate GTPase enzyme hydolyzing GTP IF1, 2, 3 initiation factor itRNA initiator tRNA H helix HSP heat shock protein LB Luria‐Bertani medium mRNA massenger RNA Nt nucleotide OB oligomer‐ binding P precursor PIC pre‐initiation complex P‐site peptidyl‐tRNA binding site PTC peptidyl transferase center RBS ribosome binding site RF1, 2, 3 release factor RRF ribosome recycling factor rRNA ribosomal RNA SD Shine‐Dalgarno sequence TIR translation initiation region tRNA transfer RNA Å Ångstöm, 1Å = 1 × 10‐10 m

8 1. Introduction

1.1 Bacterial translation process

Protein synthesis, or translation of the genetic information in mRNA (messenger RNA) into amino acid sequence of proteins, is essentially the same in all kingdoms of life and takes place on the ribosome. The ribosome which is formed of two subunits (30S and 50S subunits in bacteria) contains three binding sites for tRNA molecules: the A site binds the aminoacyl‐tRNA, the P site binds the peptidyl‐tRNA and the E site binds the deacylated tRNA. Protein synthesis can be divided into four main steps, initiation, elongation, termination and recycling. During initiation, the initiation factors (IFs) facilitate the assembly of the 30S and 50S subunits on mRNAs translation initiation region (TIR) to form an active ribosomal particle and the placement of the initiator tRNAfMet in the P‐site [2, 3]. At the end of the initiation step the A site is ready to receive an aminoacyl‐tRNA molecule which is delivered by elongation factor EF‐Tu.

The proper codon‐anticodon interactions stimulate the GTP‐ase activity of EF‐Tu leading to the dissociation of EF‐Tu from the complex. As a consequence the aminoacyl end of the A‐site tRNA releases and positions in the peptidyl‐transferase center (PTC) in a process known as accommodation. A peptide bond is then formed between the A‐ and P‐site tRNAs (α amino group of the aminoacyl‐tRNA attacks the carbonyl carbon of the peptidyl‐tRNA) at the peptidyl‐transferase center on the 50S subunit. Upon peptide bond synthesis, the lengthened peptidyl‐tRNA is bound to the A site, whereas the deacylated tRNA is in the P‐site. Peptide elongation is further promoted by the GTP‐dependent protein elongation factors EF‐G. The

GTP hydrolysis of EF‐G promotes the translocation of peptidyl‐tRNA (carrying a peptide chain one amino acid longer) from the A‐site to the P‐site and the deacylated tRNA from the P‐site to the E‐site. Consequently, the ribosome moves down the mRNA with an empty A‐site ready to receive a new tRNA molecule, the positioning of it is promoted by the elongation factor EF‐Tu

[4‐6]. Protein synthesis in bacteria terminates when a stop codon on mRNA enters the ribosomal

A‐site. Release factor 1 or 2 recognizes the stop codon and subsequently catalyses the hydrolysis of peptidyl‐tRNA, releasing the nascent polypeptide from the ribosome [7]. Release factor 3

9 triggers the dissociation of release factor 1 or 2 from the A‐site [8]. After the dissociation of RF3 from the ribosome, the ribosome must be recycled into subunits for a new round of translation initiation. Ribosome recycling factor RRF, EF‐G and IF3 proteins are required to release the deacylated tRNA, mRNA and to dissociate the ribosome subunits [9, 10]. In this study we will focus in two processes: the translation initiation and termination in bacteria.

1.2 The bacterial ribosome and its subunits

The ribosome is the largest and the most complex ribozyme found in nature. It consists of two subunits of unequal size, a small 30S and a large 50S subunit, assembles upon translation initiation and has a relative sedimentation rate of 70S. The amount of ribosomes is tightly regulated because making ribosomes is costly for the cell. The eubacteria Escherichia coli (E.coli) cell contains about 2,000 ribosomes at slow growth rate and this number can increase to 70,000 per cell during rapid growth [11].

Many cryo electron microscopy (cryo‐EM) studies have improved the structural knowledge of the ribosome and revealed new features such as the localization of several translation factors and a folded mRNA and the conformational changes associated with different functional states

[12‐15].

In E. coli, one third of the ribosome mass consists of proteins and two thirds consist of rRNA.

The small ribosomal subunit, 30S, is composed of 21 proteins and an rRNA of 1542 nucleotides sedimenting at 16S, whereas the large ribosomal subunit, 50S, is composed of 33 proteins and two rRNAs containing about 120 and 2900 nucleotides sedimenting at 5S and 23S, respectively.

The ribosomal subunits perform distinct roles during protein synthesis. The small ribosomal subunit contains the decoding center that ensures that the tRNA with the correct anticodon is bound to the ribosome and paired with the mRNA codon, whereas the large subunit contains the peptidyl‐transferase center (PTC) that catalyzes the synthesis of peptide‐bond formation [2,

16]

10 1.2.1 The small ribosomal subunit

In the small 30S ribosomal subunit, the 16S rRNA can be divided into tertiary domains which are responsible for the global shape of the small subunit (Fig 1). The 5ʹ ‐domain of the 16S rRNA forms the body, the central domain forms the platform, and the 3′ ‐domain, which is even further subdivided into one 3′ ‐major domain forms the head [17] and one 3′ ‐minor domain consists of two helices at the subunit interface, helix 44 and helix 45. H44 stretches from the bottom of the head to the bottom of the body along the 30S interface and H45 with its conserved

GGAA hairpin loop, packed against helix 44 is available for interaction with the large subunit

[18]. In the 30S particle viewed from the interface, the head is connected to the rest of the small subunit by a narrow neck which is slightly bent to the left forming a deep cleft. The platform is below the head to the right of the cleft and the decoding site with the A and P sites is located at the bottom of the cleft between the head and the body [17, 19]. The decoding center, where the codon‐anticodon interaction takes place, is entirely constructed of rRNA. This region contains the 3′ and 5′ ends of the 16S rRNA and the upper part of helix 44 [19]. Recently, two more regions with specific activities in translation have been described. The first one is the platform center which plays an important role in the binding and adaptation of the mRNA during translation [20, 21]. The other one is the helicase center, constituted by proteins S3, S4 and S5, devoted to the unfolding of structures at the 3′ end of mRNA during elongation [22, 23].

The 30S ribosomal proteins are concentrated in the top, sides and back but none of them binds entirely inside an RNA domain. The subunit interface, where the interaction with the large subunit occurs, is free of protein, with exception of protein S12 which lies near the decoding site at the top of H44. Some other proteins lie at the periphery of the subunit interface which allow them to make contact with the 50S subunit [18] (Fig 1A).

1.2.2 The large ribosomal subunit

In the large 50S ribosomal subunit, the secoundary structure of 23S rRNA can be subdivided into six domains [24] and the 5S rRNA is considered as the subunits seventh rRNA domain [25].

It is important to note that the secondary‐structure domains of the rRNA constitute distinct

11 morphological domains of the intact 30S subunit but not for the 50S subunit [17]. The 5S rRNA and the six secondary structure domains of 23S rRNA all have complicated and convoluted shapes that fit together to produce a compact, monolithic RNA mass and the 5S and 23S rRNA do not interact extensively with each other [26, 27]. When the 50S viewed from the interface side, three protuberances can be shown. The central protuberance (CP) which include the 5S rRNA and its associated proteins, the L1 stalk, consisting of protein L1 and its 23S rRNA binding site and the L7/L12 stalk, which includes the L11 arm, consisting of protein L11 and its

23S rRNA binding site [28].

Figure 1. Tertiary structures of the 30S (A) and 50S (B) subunits, seen from the interface side

Ribosomal proteins are shown as blue ribbons and rRNA is shown as translucent gray spheres.

2007, American Society for Microbiology

The central enzymatic activity of the large subunit, the peptidyl‐transferase center (PTC), is located in Domain V. There are 15 proteins that interact with this domain but no protein moiety is observable within about 18Å of the PTC which means that the ribosome is a ribozyme [30].

The polypeptide exit tunnel begins just below the PTC and provides a stable passage of the nascent polypeptide through the subunit to the cytoplasmatic side of the 50S subunit. This tunnel is approximately 100Å long and up to 25Å in diameter and largely formed by RNA [30].

12 Most of the 50S ribosomal proteins are located on the external surface whereas the interior is protein‐poor [26, 27] (Fig 1B).

1.3 Assembly of ribosomal subunits

Ribosome synthesis and assembly is tightly controlled process because the finished ribosomal subunits must function perfectly to guarantee efficiency and quality in protein synthesis. The assembly of ribosome occurs in a series of steps: rRNA maturation and modification, binding of ribosomal proteins and rRNA folding and the binding and release of ribosome’s assembly factors. Many of these steps are coupled and occur in parallel during the transcription of rRNAs [31, 32].

1.3.1 rRNA maturation and modifications

Ribosomal RNA (rRNA) is transcribed as a single transcript including both the 16S rRNA for the small subunit and the 23S and 5S rRNAs for the large subunit separated by spacer tRNAs and some extra sequences such as the complementary sequences flanking both 16S and 23S rRNA to form strong base paired stems (Reviewed in [29]) (Fig 2). The synthesis of rRNA is highly regulated by a so called feedback regulation [11]. The single primary transcript is processed into mature 5S, 16S, and 23S rRNA by at least five nucleases and starts even before transcription of the ribosomal RNA is completed [33‐36].

RNase III cleaves the primary transcript to yield precursor 16S rRNA (17S rRNA), precursor

23S rRNA, and precursor 5S rRNA (9S rRNA) which contain the sequences for the mature 16S,

23S, and 5S rRNA respectively [37‐39]. 17S rRNA contains the 16S rRNA sequence with an additional 115 nucleotides (nt) at the 5ʹ end and 33 nt at the 3ʹ end [37]. The 5ʹ end is further cleaved by RNase E to yield a product with 66 extra nucleotides at the 5ʹ end, which is subsequently cleaved by RNase G [40]. Maturation of the 5ʹ end occurs before the 3ʹ end [40, 41] and can occur in the absence of RNase E but more slowly [40]. The final processing of the 3ʹ end to remove the extra 33 nucleotides is performed by an unknown RNase and this reaction occurs efficiently under protein synthesis conditions [42]. Interestingly, mature 16S rRNA can still form

13 in the absence of RNase III; however mature 23S rRNA cannot be formed [36]. The 23S rRNA

precursor formed by RNase III cleavage contains the mature 23S rRNA transcript with three or

seven additional nucleotides at the 5ʹ end and seven to nine additional nucleotides at the 3ʹ end

[35, 43]. Final processing of the 5ʹ end of precursor 23S rRNA is performed by still unknown

enzyme, while final maturation of the 3ʹ end facilitated by RNaseT [44] (Fig 2). Following

cleavage by RNase III, the 3ʹ ‐terminal part of the primary transcript contains 5S rRNA and

additional sequences that may include one or two distal tRNAs. The 5′ ‐termini of the tRNA

sequences are processed by RNase P which results in the release of 9S rRNA [45]. 9S rRNA

contains the mature 5S rRNA with additional 84 nt at the 5ʹ end and 42 nt at the 3ʹ end [46].

RNase E is able to cleave the precursor at both ends to leave mature 5S rRNA and three

additional nucleotides at both ends [47]. RNase T causes final maturation at the 3ʹ end [48]

while final maturation at the 5ʹ end occurs by a still unknown enzyme (Fig 2).

5′ 3′ Figure 2. Typical rRNA operon, shown schematically. The spacer region with tRNA species

between the genes for 16S, and 23S rRNA are shown. Promoters P1 and P2, and terminators T1

and T2 are also indicated. The cleavage site of RNase III (III), RNase G (G), RNase E (E), RNase

P (P), RNase T (T), and the unknown RNases (?) are shown. From [29].

Most of the modifications in both 16S and 23S rRNA occur on conserved nucleotides (nt) that

are located on functionally important regions [49, 50]. These modifications are thought to

influence the structure and the function of the ribosome. 16S rRNA is known to undergo 11

14 modifications, of which 10 are methylations and one pseudouridine, and 23S rRNA undergoes

25 modifications, of which one is unknown, one is a methylated pseudouridine, 14 are methylations and 9 are pseudouridines [29, 50, 51]. Remarkably, some modifications of the 16S rRNA are added to naked rRNA while others are added during late maturation of the 30S rRNA whereas the modification of the 23S rRNA is mainly an early event [29]. Moreover, unmodified in vitro transcribed 16S rRNA with all of the natural 30S ribosomal proteins can reconstitute 30S particles with somewhat reduced tRNA‐binding capacity compared to 30S subunits reconstituted with rRNA modified in vivo [52]. Unlike the small subunit, in vivo reconstitution of a catalytically active large ribosomal subunit seems to depend more on chemical modification of 23S rRNA in E.coli [53]. It was has been shown that the 23S rRNA modifications that are most important for in vitro reconstitution of catalytically active particle are located in region of 80‐nt (nt 2445‐2523) that contains 7 of the 25 known modifications [53].

In vivo modifications that take place outside of the 80‐nt region, like the three ψ in helix 69

(1911, 1915 and 1917) and Um2525, may also be important [54, 55]. The importance and role of each modification remains obscure; it has been shown that some modifications may alter the features of the nucleotides providing an additional way to fine‐tune the rRNA folding and its interaction. It has also been suggested that methylation, for instance could modulate rRNA maturation and affect stability of rRNA structures. One of the possible functions of the modification could be to act as structural checkpoint (reviewed in [29]).

1.3.2 Binding of ribosomal proteins and rRNA folding

In vitro reconstitution studies of E. coli 30S and 50S ribosomal subunits [56, 57] have established the knowledge of how these individual protein and RNA molecules come together to form complete and functional ribosome. The studies showed that an active ribosomal subunit could be assembled in vitro simply by incubating the mature components together under appropriate conditions [56, 57]. This means that all the information required for the proper ribosome assembly is present in the ribosomal rRNA and proteins themselves [56, 57].

However, in vitro reconstitutions of ribosomal subunits occur more slowly than in vivo and the

15 reconstitution of the 50S subunit is comparatively more slow and inefficient [58, 59] and as a result has not been studied extensively (see review [60] and reference therein). The first precursors, the RI30 and RI50 are formed at 0°C upon incubation of a subset of proteins and 16S or 5S and 23S rRNA respectively. Reconstitution can not continue until the particle is heated to

∗ ∗ 40°C to rearrange the conformation and form more compact particles RI30 and RI50 . This change allows the association of further proteins to form the active 30S subunit, while the formation of active 50S subunit requires continued incubation with proteins, followed by a second heating step at 55°C with high magnesium concentration [56, 57]. It was also shown that in vitro reconstitution of the 30S subunit requires less energy if the latter is assembled from precursor instead of mature rRNA [61], suggesting that proper processing of the precursor 16S rRNA plays a key role in guiding certain proteins to their appropriate binding sites and in accelerating the process (Reviewed in [29])

Figure 3. The Nomura assembly map depicts thermodynamic protein binding dependencies in the 30S subunit. There is a clear hierarchy of primary binding proteins (1°), which stably bind directly to the rRNA; secondary binding proteins (2°), which depend on primary binders; and tertiary binding proteins (3°), which depend on secondary binders. The map is further divided into 5′ (red), central (green), and 3′ (blue) domains on the basis of binding position relative to the 16S rRNA [62‐64]. From [60]

Nomura and coworkers showed that the protein‐binding events are thermodynamically interdependent and that the ribosomal proteins bind to 16S rRNA in a hierarchical manner [56].

Nomura and colleagues work resolved the complete assembly map for the 30S subunit [62, 63].

The original map by Nomura has been further divided into 5, central and 3 structural domains on the basis of binding proteins relative to the 16S rRNA [64]. From the assembly map the 30S ribosomal proteins can be classified into three groups according to their physical location and order in the binding hierarchy: primary binding proteins, can bind directly to 16S rRNA, secondary binding proteins, require prior binding of one of the primary proteins and tertiary binding proteins that need at least one primary and one secondary protein for binding [62, 63,

65, 66]. Powers et al suggested that the assembly proceeds mostly from the 5′ to the 3′ end of

16S rRNA [67] (Fig 3).

Herold and Nierhans developed thereafter a similar assembly map for the 50S subunit [68], but the assembly is not organized by structural domains and many more proteins associate in a more complex binding hierarchy. The experimental approaches to the 50S subunit assembly are reviewed by [69] but not discussed here.

The exact mechanism by which ribosomal proteins and rRNA organize themselves remains unknown. The hierarchy of protein binding leads to cooperative assembly and this cooperativity mostly arises from structural changes in the 16S rRNA induced by the binding of previous protein [33, 70]. The assembly process appears to occur by an alternating series of

RNA conformational changes and protein‐ binding events. A local RNA folding event creates a protein binding site, which in turn binding of each facilitates the next RNA folding event and incrementally driving the RNA structure to the final native state [31, 32]. However, the ability of

5ʹ domain of the 16S rRNA to form all of the predicted tertiary interaction in the absence of proteins indicates that the initial step in RNA folding is driven purely by the RNA [71]. And ribosomal proteins play two potential roles in the assembly process, guiding the rRNA into the proper conformations and stabilizing the native secondary structure. For instance the small subunit ribosomal protein S12 has been shown to have RNA chaperone activity in vitro [72] and

17 in vivo [73]. Footprinting studies showed that binding of S4, S20 and S17, primary binding proteins, preorganize the binding site for S16, a secondary binding protein, and binding of S16 directing the assembly toward the native folded state by destabilizing the structure of non‐ native conformation [74, 75]. Moreover, it has been shown that about one third of the 34 large

50S ribosomal subunit proteins exhibit RNA chaperone activity [76].

Most ribosomal proteins are essential for cell viability like the 5S rRNA binding proteins L5 and L18 [77]. However some nonessential ribosomal proteins can still convey a selective advantage in growth rate, for instance a ribosome that lacks ribosomal protein L25 is viable and functional but displays a slow growth phenotype [77], or can be important for efficient assembly and translation capacity like protein S20. A ribosome that lacks ribosomal protein S20 is viable but loses the ability to form the 70S ribosome [78, 79]. Besides the ribosomal proteins, there are other proteins called ribosome assembly factors, which guide the rRNA into the proper conformation and stabilize the native structure of the RNA. These factors may serve as sensors of checkpoints during the assembly process. These assembly factors are especially important for assembly of the more complex 50S subunit (see the section down).

1.3.3 Ribosomal assembly factors

Proper rRNA folding is one of the most complicated events during ribosome assembly because the RNA molecule can easily form distinct secondary structures and both the number and the stability of these non‐functional structures increase with the growing length of the RNA

[80, 81]. Many of these alternative secondary structures result in kinetically trapped intermediates that reach the native structure very slowly and this can explain why the assembly process is much slower in vitro than in vivo [82, 83]. In the cell, ribosome assembly factors allow the assembly process to progress more quickly by preventing these kinetic traps and facilitating proper rRNA folding and Protein‐RNA interactions. These factors may also lower the activation energy required for maturation and thereby omit the heating step that is required to complete the maturation in vitro [33] (see section1.3.2 ). Such assembly factors that facilitate and speed‐up

18 ribosome assembly include DEAD‐box proteins, GTPases, chaperones and maturation factors.

Some of these factors will be discussed here.

DEAD‐box proteins

DEAD‐box proteins are a large family of RNA helicases [84] that poses RNA‐dependent

ATPase activity [85]. DEAD‐box proteins and the related family DExD/H are believed to have multiple roles in ribosome assembly. They help unwinding of local RNA secoundary structures and assist proper RNA folding, rearranging or dissociating RNA‐protein interactions [86, 87].

The E.coli ribosome has been associated with three members of the DEAD‐box helicase family

(SrmB, CsdA and DbpA) [88‐92].

The DEAD‐box protein SrmB is thought to be involved in the early step of 50S biogenesis because a strain with a deletion in the srmB gene has fewer 70S particles, an increased number of free subunits as well as accumulation of a 50S precursor particle that sediment at approximately 40S. Moreover, SrmB can associate with the 40S precursor particle, but not with

30S, 50S subunits or 70S particles. This 40S particle contains immature 23S rRNA and has a reduced amount of some ribosomal proteins or missing some other like protein L13 [90], essential for the formation of the first 50S intermediate in vitro [93].

The cold‐shock DEAD‐box protein A, CsdA, is required for growth at temperatures below

30°C and deletion in the csdA gene leads to a slow growth at low temperature [94] and to accumulation of the 50S precursor particles that sediment at approximately 40S [91].

DEAD‐box protein A, DbpA, is an ATP‐dependent helicase [95, 96]) and deletion in the dbpA gene does not result in a growth defect [97]. A strain with a mutant DpbA has an increased number of free subunits, fewer 70S particles, as well as accumulation of a 50S precursor particles that sediment at approximately 45S, implying that the DpbA plays a role during the late stages of ribosome assembly [92].

GTPases

GTPases is another class of proteins that have been proposed to be involved in the assembly of the ribosome, mainly in the biogenesis of the individual subunits. These proteins have

19 additional domains that are expected to mediate the interaction of the GTPases with the ribosome through direct binding to rRNA, binding to a ribosomal protein, or both [98, 99].

Era (E. coli ras) is one of the highly conserved, essential GTPase that binds to 16S rRNA and the 30S ribosomal subunits in vitro [100]. Era binds to the 30S subunit between the head and cleft on the side of the subunit that interacts with the 50S subunit to form a 70S ribosome [101].

This finding suggests that Era would prevent subunit joining prior to the final maturation of the

30S subunit into an active particle. Depletion of Era leads to the accumulation of precursor 16S rRNA and an increase in the free 30S and 50S subunits compared to the 70S particles implying that Era may has a role in the processing of rRNA [102]. It has been shown recently that Era enables faster binding of several late‐binding proteins to rRNA when included in a 30S reconstitution [103].

Chaperones and maturation factors

Another group of RNA‐binding proteins that can help solving the RNA folding problem are called chaperones [104]. The chaperones DnaJ and DnaK are part of the heat shock protein 70

(HSP 70) chaperone machine DnaK‐ DnaJ‐CrpE [105]. Deletion of the dnaJ or dnaK genes leads to assembly defect at a temperature above 42°C with accumulation of 30S precursor that sediment at 21S and 50S precursors that sediment at 32S and 45S [41]. At this temperature over‐ expression of heat shock proteins GroEL‐GroES can partially restore this defect [41, 105], suggesting an overlapping function between DnaK‐ DnaJ‐CrpE and GroEL‐GroES. Recently it has been shown that one of the translation initiation factors, IF1 can posses rRNA chaperoning activity [106].

Finally, assembly of ribosomes is facilitated by some other proteins called maturation factor.

Ribosome‐binding factor A (RbfA) is one of these factors that is induced upon cold shock [107].

RbfA has been shown to associate with free 30S subunits and cells lacking RbfA are cold sensitive, have impaired growth rate and have ribosome profile defect [108]. Strain with a deletion in the rbfA gene has an increased amount of 16S precursor [109], implying that RbfA can be a late maturation factor that is essential for efficient processing of 17S rRNA to 16S rRNA. That RbfA interacts with the 5′ ‐terminal helix region of the 16S rRNA, a helix that is

20 found in mature 16S rRNA, indicating that RbfA may also play a role in maturation after the formation of 16S rRNA [110]. Ribosome maturation factor M (RimM) is also essential for efficient processing of the 16S rRNA because a deletion of rimM leads to the accumulation of precursor 16S rRNA and an increase in the free 30S and 50S subunits [110].

1.4 Bacterial translation initiation

During translation initiation, the rate‐limiting and the most regulated step, the ribosomes assemble on the mRNA in the order of seconds [111]. The three initiation factors are required to enhance the fidelity and accuracy of the initiation process and to speed up the formation of the

70S initiation complex (70SIC) [112]. The initiation process begins at the level of the 30S that is dissociated from the 50S subunit by the action of IF3 which is assisted by IF1. Subsequently binding of IF2 to the 30S subunit assist the binding of the initiator fMet‐tRNAfMet and the translation initiation region (TIR) of the mRNA to the ribosomal subunit forming a 30S preinitiation complex (Reviewed in [3]). Next a short duplex between the SD sequence of mRNA and the anti SD (aSD), a conserved sequence at the 3′ end of 16S rRNA is formed [113].

The mRNA is then accommodated, with the help of the IFs and the fMet‐tRNAfMet, into the mRNA channel leading to the formation of more stable and active 30S initiation complex. In this complex the start codon of the mRNA interacts with the anticodon of the initiator tRNA in the ribosomal P‐site [2, 114‐116]. The last step of initiation process involves the formation of the 70S initiation complex (70SIC) by docking of the large ribosomal subunit (50S) subunit to the 30SIC.

The docking process activates the ejection of IF1 and IF3 from the ribosome. This transition triggers the hydrolysis of the GTP molecule bound to IF2 and results in the adjustment of the initiator fMet‐tRNA in the P‐site and IF2 dissociates from the ribosomal complex [117]. The first peptide bond is formed in an EF‐Tu GTP‐dependent step specified by the second mRNA codon and the translation enters the elongation phase [5, 118].

21 1.4.1 The translation initiation region in messenger RNA

The most critical event in translation initiation involves the recognition and the binding of the 30S to the translation initiation region (TIR) on mRNA in order to select the appropriate initiation codon. The prokaryotic translational initiation region (TIR) includes the ribosome‐ binding site (RBS) and the bases extending beyond 5′ and 3′ of the (RBS) [2]. The (RBS) comprises the Shine‐Dalgarno sequence complementary to a conserved sequence present at the

3′ end of 16S rRNA [113] and the start codon plus a spacer of variable length, separating the SD sequence and the initiation codon [115, 119]. The sequence upstream of the initiation codon is called the 5′ untranslated region (5′ UTR) and it includes the SD sequence and AU‐rich sequences upstream of SD act as the recognition motif for ribosomal protein S1 and shown to enhance translation in E.coli with a weak SD [120‐122]. AUG is the most common initiation codon and used in 90% of bacterial genes [123, 124]. The spacer region between the SD and the start codon, which varies between 3 and 12 nucleotides with an optimal spacing of 7‐9 nucleotides, has been shown to influence the efficiency of translation initiation [125, 126]. The downstream region (DR) following the initiation codon has been proposed to enhance translation of several mRNA (for a review, see [127]). The mechanism proposed for the stimulation of translation is through a complementary base pairing between the DR and the nucleotides 1469‐1483 in helix 44 of the 16S rRNA [128, 129]. However, many studies failed to support this proposal [130‐132]. A recent study shows that Shine‐Dalgarno like sequences in the downstream coding region affect the translation initiation negatively, more likely by guiding the ribosomes away from the start codon [133].

1.4.2 The initiator transfer RNA (itRNA)

The bacterial initiator tRNAfMet has a special function in the cell; it reads the start codon aligned in the P‐site, in contrast with elongator tRNAs which binds first to the A‐site of the 30S subunit, allowing the initiating ribosome to begin translation in the correct location [3]. Binding of tRNAfMet to the P‐site is facilitated by initiations factors IF1, IF2 and IF3 [119, 134].

22 Several structural features distinguish the initiator tRNA and suggests a mechanism by which the translation initiation machinery can discriminate the initiator tRNA from elongator.

Initiator tRNA contains a C:A mismatch at position 1:72 in the acceptor stem, which has been shown to be a determinant for formylation of the methionine moiety by methionyl‐tRNA transformylase [135, 136]. The presence of formylmethionine is also one of the important features for binding of the fMet‐tRNAfMet to IF2 [137]. The second feature is the presence of the three conserved G‐C base pairs in the anticodon stem of the initiator tRNA that confers a specific IF3 selection of the initiator tRNA to the P‐site [138]. It was also shown that there is a minor groove interactions between A1339 and G1338 of the 16S rRNA and the G‐C base pairs

30‐40 and 29‐41 which may play a role in the discrimination of the initiator tRNA by IF3 and provide stabilization for the initiator tRNA in the P‐site [139‐141]. A recent study has revealed the crystal structure of E.coli initiator tRNA at 3.1Å resolution. In this study the length of the anticodon stem was extended by one base pairs Cm32‐A38 and an unique structure of the anticodon loop that involves a base triplet between A37 and the G29‐C41 pair was also revealed

[142]. The third feature of the initiator tRNA is the presence of a purine 11: pyrimidin 24 base pair in contrast to pyrimidin 11: purine 24 in other tRNAs [143].

Transfer RNA modification

Transfer RNA from all organisms contains modified nucleoside derivatives of the four normal nucleosides adenosine (A), guanosine (G), uridine (U), and cytosine (C) [144, 145]. The presence of different modified nucleosides is likely to have different effects on the activity of tRNA; it may influence the efficiency of the tRNA in the decoding steps. Thus, lack of a modified nucleoside may induce pleiotropic effects on cell physiology (reviewed in [145]). The positions of the modified nucleosides have become known and a number of them are located at specific positions on tRNAs demonstrating one of the characteristics of tRNAs [146]. For instance, the wobble position (position 34), the first position of the anticodon, is often modified

[146]. In bacteria the uridine at position 34 in tRNALys, tRNAGlu, and tRNAGln is often modified to 5‐methylaminomethyl‐2‐thiouridine mnm5s2U34 [147] and the sulfur in the thiolated nucleoside originates from cysteine [148]. The effect of 2‐thiouridine s2U‐34 modification on

23 stabilization of tRNA codon‐anticodon interactions has been noted, 2‐thiouridine s2U have almost a conformation that allows the recognition of A but not U and G in the third position of the codon [149]. It has been shown that tRNAs with s2U34 modification instead of mnm5s2U34 are more able to read UAA than UAG codons [150]. Moreover, the efficiency of tRNAUAALys in reading both UAA and UAG in an E.coli strain defective in the synthesis of mnm5s2U34 is also reduced [151]. Thus, the modification of this position and the modification of tRNA in common seem to optimize tRNA to decode mRNA efficiently and accurately (reviewed in [145]). Björk and Hagervall showed a model for how the deficiency in tRNA modification, hypermodification, or an otherwise defective tRNA can induce frameshifting + 1 in the P‐site

[152]. The model suggests that defects in tRNA may induce framshifting at two important steps in translation either by affecting the rate at which the ternary complex binds to the ribosomal A‐ site, A‐site effect, or by altering the interaction of the peptidyl‐tRNA in the ribosomal P‐site, P‐ site effect [153]. Finally, Agris P et al showed that the 2‐thiouridine s2U modification is required for binding of tRNAUUULys to programmed ribosomes, possibly because of a structural alteration in the anticodon loop. In other words, the fact that the unmodified tRNALys did not bind well to the ribosome in their assay is a further evidence for slippage and frameshifting as suggested by

Björk and Hgervall [152] and which we suggest as the best explanation for why the lack of the

2‐thiouridine s2U modification is required for suppression of the Ts phenotype caused by mutant RF1 as discussed in (Paper III).

1.4.3 Initiation factor 1 (IF1)

IF1 is the smallest initiation factor with a molecular mass of around 8,2 kDa in E.coli. IF1 is an essential protein, which participates in the translation initiation process in prokaryotes and it is homologous to the aIFA and eIF1A proteins in archea and eukaryotes, respectively [154, 155].

The structure of IF1, determined with multidimensional NMR spectroscopy [156], is characterized by a rigid five‐stranded β‐barrel flanked by the N‐and C‐terminal tails which are disordered and highly flexible [156]. The IF1 fold shows similarities to proteins that interact with oligosaccharides and oligonucleotides like proteins belonging to the oligomer‐ binding

24 (OB) family, and to so call cold shock proteins CspA, CspB [156‐159]. The same RNA‐binding motif has been found in the 30S ribosomal protein S1 [160, 161]. The crystal structure of the 30S‐

IF1 complex at high resolution 3,2Å shows that IF1 bound to the 30S subunit is in proximity to the A‐site [162] in a cleft formed between helix 44, the 530 loop and protein S12 (Fig 4). IF1 also interacts with functionally important bases A1492 and A1493 of the 16S rRNA helix 44, causing these two bases to flip out, and tilting the head of the subunit towards the A‐site [163].

Mutagenesis of these two nucleotides was found to inhibit IF1 binding [164].

IF1 530 loop Helix 44 Head Platform

Body S12

Figure 4. The binding site of IF1 in the 30S subunit. A) The interaction of IF1 with the 30S subunit. Important features are labeled. B) The location of IF1 shown from the interface side of the 30S subunit. Picture adapted with permission from [163]. Copyright © 2001, AAAS.

Several functions have been attributed to this factor, aside from promoting the binding efficiency of IF2 and IF3 to the 30S subunit, IF1 cooperates with IF2 to ensure the correct positioning of the initiator tRNA in the P‐site [165, 166] and discriminate together against unformylated and deacylated tRNA [167, 168]. IF1 also stimulates the GTPase activity of IF2 and increases binding of mRNA in the presence of IF2 [169, 170]. It was shown that IF1 is

25 needed for IF2 recycling after subunit joining and GTP hydrolysis [171]. Interestingly, recent data show that IF1 can behave as RNA chaperone in vivo and in vitro [106] and as a transcriptional antiterminator in vivo [172]. IF1 was also suggested to stimulate the 30S initiation complex formation by inducing a conformation change over a long distance in the small ribosomal subunit [166] and thus influencing the association‐dissociation equilibrium of the ribosomal subunits [173, 174]. This function is a focus of the paper I in this thesis.

1.4.4 Initiation factor 2 (IF2)

Translation initiation factor 2 (IF2) is the largest of the three factors involved in initiation of protein biosynthesis in eubacteria and belongs to the family of the GTP‐GDP binding proteins like the elongation factors EF‐Tu and EF‐G [175]. The bacterial initiation factor IF2 consists of three major segments: the less conserved N‐terminal region, the highly conserved G domain and the C‐terminal part [176, 177]. The N‐terminal region is proposed to enhance the interaction of IF2 with the 30S and 50S ribosomal subunits [177, 178]. The C‐terminal part contains the fMet‐tRNA binding domain and is responsible for the recognition of the formylated form of fMet‐tRNA [179‐182], while the G domain contains the GTP binding site [177, 183]. The N‐ terminal and central segments of the IF2 protein were found to be connected to the C‐terminal domain via a long and flexible linker [184].

IF2 promotes codon‐anticodon tRNA pairing [168, 185, 186] and plays an important role in the docking of 50S subunits to 30S PICs containing the fMet‐tRNA but not un‐formylated fMet‐ tRNA or elongator tRNA [168, 187]. It has been also reported that the GTP‐bound form of IF2, but not GTP hydolysis, promotes rapid docking of the 50S subunit to the fMet‐tRNA‐contiaing

30S PIC [188, 189]. A recent study shows that, two classes of mutations located outside the tRNA‐binding domain of IF2 could compensate strongly, A‐type, or weakly, B‐type, for initiator tRNA formylation deficiency [187]. More recently, the same group showed that A‐type IF2 mutants, but not wild‐type IF2, bypass not only the formylation requirement but also the requirement of an initiator tRNA bound in the P‐site for rapid docking of ribosomal subunits.

Moreover, A‐type IF2 mutants with either GTP or GDP can promote rapid subunit docking,

26 implying that fMet‐tRNAfMet, per se does not have an active role in rapid subunit docking.

Instead, initiator tRNA is essential for activation of wild‐type IF2 in the 30S PIC and thus drives the 30S PIC to active 50S subunit‐docking conformation [190].

1.4.5 Initiation factor 3 (IF3)

Bacterial initiation factor IF3 is one of the three initiation factors required for efficient translation initiation [3]. E. coli IF3 is an essential protein composed of 180 amino acids with a molecular mass of 20,4 kDa [191, 192]. IF3 contains two structural domains of approximately equal size; the N‐ and C‐terminal domains [193, 194] which are joined by a long, flexible lysine‐ rich linker [195, 196]. This linker has been shown to be essential for IF3 function [197]. The C‐ terminal domain is thought to be responsible for 30S subunit binding while the N‐terminal domain does not bind to the 30S on its own but appear to contribute to IF3 binding stability

[198‐200]. A single amino acid substitution in the N‐terminal domain has been shown to impair all the known functions of IF3 in vivo [201]. A recent cryo electron microscopy (cryo‐EM) study shows that the N‐terminal domain of IF3 contacts the tRNA, whereas the C‐terminal domain binds to the platform of the 30S subunit [15].

Several important functions have been attributed to IF3 [200]. It binds to the small 30S ribosomal subunit and prevents the association between the 30S and the 50S subunit [202]. IF3 promotes the correct 30S initiation complex formation by stimulating the codon‐anticodon interaction between the fMet‐tRNAfMet and mRNA in the P‐site [165, 203]. Moreover, IF3 affects the initiation fidelity when it preferentially dissociates preinitiation complexes with aminocyl‐ tRNA (non‐initiator tRNA) [167, 204], complexes with non‐canonical start codons [205‐207] and complexes containing leaderless mRNA [208]. Both IF3 domains have been shown to be involved in these functions because mutation in either the N‐ or C‐ terminal domain can perturb

IF3 role in start‐codon discrimination [209, 210]. IF3 is also involved in shifting the mRNA from the standby site to the decoding P‐site of the 30S ribosomal subunit [211]. Finally, IF3 enhances dissociation of the deacylated tRNAs from posttermination complexes and dissociates the 70S subunits during ribosome recycling [10, 212].

1.4.6 The antibiotic kasugamycin as a translation initiation inhibitor.

The key role of the bacterial ribosome makes it the site of action for many antibiotics [213].

Kasugamycin is an aminoglycoside antibiotic [214] that blocks translation initiation in bacteria

[215]. Kasugamycin is known to prevent binding of initiator fMet‐tRNA to the P‐site on the 30S subunit and on 70S ribosomes [215]. X‐ray structure of kasugamycin bound to the 30S subunit

[216] or to the E.coli 70S ribosome [217] reveals the kasugamycin binding site within the messenger RNA channel in the P and E sites of the small ribosomal subunit (Fig 5).

Kasugamycin seems to interact with two universally conserved A794 and G926 nucleotides of the 16S rRNA [218] which is consistent with the fact that kasugamycin resistance arises when these two nucleotides are mutated [219]. The above mentioned evidence about the position of the kasugamycin binding site [216, 217] can explain why the antibiotic can block different steps of translation initiation (as discussed in paper II).

In early studies, it was shown that a mutation in the ksgA gene, responsible for dimethylation of A1518 and A1519, confers resistance to the antibiotic kasugamycin [220], increases decoding errors during elongation [221], enhances translation initiation from non‐AUG codon [222], and exhibits cold sensitive 30S assembly and 16S processing defects [223]. More recently, X‐ray analysis has indicated that the dimethylation of these two adenosines A1518 and A1519 in the

GGAA tetraloop of the 16S rRNA H45 at a late stage of 30S subunit assembly [224] facilitates structural rearrangements in order to establish an active conformation of the 30S subunit and optimize it to participate in protein synthesis [225]. All these data suggest that the kasugamycin may affects a step during subunit joining or later [217]

The effect of kasugamycin does not operate by completely preventing the binding of mRNA to the ribosome. However, kasugamycin perturbs the conformation of mRNA and influence positioning of the mRNA at the P‐ and E‐sites within the mRNA pathway, thus preventing efficient fMet‐tRNA binding [216, 217, 226]. Thereby, translation initiation of leaderless mRNAs, starting directly with 5´ AUG, is not inhibited by kasugamycin [227, 228]. It was shown that the inhibitory effect of kasugamycin on translation can be different depending on different mRNA

28 [229] and this inhibition is dependent to some extent on the E‐site triplet [217]. It was also shown that kasugamycin, unlike other aminoglycosides, does not induce translational misreading [230, 231], readthrough or frameshifting [232], however increased translation fidelity in some cases were observed [233]. More recently, kasugamycin has been shown to increase expression of reporter genes that are translation initiation region (TIR)‐dependent, and that kasugamycin apparently share an identical mode of action with the cold‐sensitive IF1 mutant [234]. (This similarity was investigated in paper II)

Head

Platform

Body

Figure 5. The structure of ksg bound to the E.coli 70S ribosome determined by X‐ray crystallography. Location of the Ksg‐binding pocket in the context of the 30S subunit, Ksg is shown in cyan, the 790 loop in green, the 926 region in blue and the DASL in red. Residues

29 A794, G926, A1518 and A1519 are shown as sticks. Picture adapted with permission from [217].

1.5 Bacterial translation termination and recycling

Translation in bacteria terminates when a stop codon on mRNA reaches the decoding center in the A‐site of the small ribosomal subunit. Three stop codons UAA, UAG and UGA signal that the nascent polypeptide should be released from the ribosome. These stop codons are decoded by protein factors called class 1 release factors (RF) (Fig 6) [235‐237]. In bacteria the UAA and

UAG stop codons are recognized by release factor RF1, and the UGA and UAA by RF2 [238‐

240]. Once stop codon recognition occurs, class I release factor RFs stimulate hydrolysis of the ester bond that links the completed polypeptide chain with the tRNA in the P‐site, resulting in the release of the polypeptide chain from the ribosome [239, 241, 242]. Subsequently, the release of the bound class I release factor from the ribosome is facilitated by a class II release factor RF3 in a GTP dependent manner [8]. Zavialov et al suggested that ribosome‐class I release factor complex enhance the binding of GTP to RF3 only after the release of the polypeptide chain

[243]. This in turn leads to a conformation change in RF3 and the dissociation of class I release factor. After the dissociation of class I release factor from the ribosome, a GTP hydrolysis by

RF3 results in its own release.

Finally, and in order to reuse the ribosome and the tRNA for the next round of protein synthesis the translation complex needs to break down. This process requires three proteins;

IF3, ribosome recycling factor (RRF) and EF‐G. RRF and EF‐G catalyzes the dissociation of ribosomes into subunits in a reaction requiring GTP hydrolysis. This leads to a complex in which the 30S subunit remains bound to the mRNA with a deacylated tRNA in the P‐site. IF3 activates the dissociation of the tRNA from the 30S subunit allowing it to recycle [10, 244, 245]

1.5.1 Class 1 release factors

After the discovery of release factors (RF), many questions concerning RF function and the mechanism of translation termination were needed to be resolved. The crystal structure of the isolated RF1 from Thermotoga maritime at 2,65 Å [246] and RF2 from E. coli at 1,8 Å [247] showed

30 that the factor is composed of four domains. The overall fold of the four domains was similar between the two factors, except that the RF1 N‐terminal domain is shorter and the C‐terminal domain is longer than that of RF2 (Fig 6A) [246].

Figure 6. A) Comparison of the structures of the RF1 and RF2 in its ribosome‐bound conformation rotated ≈ 180° from the view shown in B, with domains numbered. The GGQ and

PVT are shown in red, the domains 1‐4 are indicated, and the switch loop is shown in orange, B)

The structure of the RF1 termination complex, showing RF1 (yellow), P‐site tRNA (orange), E‐ site tRNA (red), mRNA (green), 16S rRNA (cyan), 23S and 5S rRNA (gray), 30S proteins (blue), and 50S proteins (magenta). Picture adapted with permission from [248] and [249]. Copyright ©

2008, Rights Managed by Nature Publishing Group.

31 The element of release factor involved in stop codon specificity recognition was localized in domain 2, in particular, to the conserved motif PxT (ProXxxThr in RF1) or SPF (SerProPhe in

RF2). Genetic experiments have shown that swapping these motifs of RF1 and RF2 led to switch codon specificity [250, 251]. At the other end of the protein, a universally conserved GGQ

(GlyGlyGln) motif in domain 3 in RF1 and RF2 [252] was proposed to be essential for promoting the hydrolysis of the peptidyl‐tRNA ester linkage [252‐257]. In cryo‐EM reconstructions and X‐ray crystal structures of termination complex, RF1 and RF2 were found to occupy the A‐site in the ribosome with domain 2 close to the stop codon and the GGQ pointed into the peptidyl‐transferase center PTC (Fig 6B) [258‐261]. Recently, more details insights into translational termination come from the high resolution crystal structure of RF1 or

RF2 bound to the ribosome [248, 249, 262]. The structures reveal how stop codons are recognized by RF1 and RF2 and how the stop codon recognition is communicated to PTC. The structures showed that domain 2 is involved directly in the recognition of the bases in the stop codons and the universally conserved GGQ motif of domain 3 contacts the acceptor end of the

P‐site tRNA, positioning the glutamine backbone to contribute directly to peptidyl‐tRNA hydrolysis [248, 249, 262]. Substitution of glutamine in this position by proline abolishes the catalytic activity of the factor [249]. The factor in complex with the ribosome seems to undergo global conformational changes and these conformation changes are accompanied by rearrangements of specific regions. Domain 3 shifts considerably from domain 2 and 4 which interact with the decoding center and domain 1 shifts slightly and interacts with the L11 region of the 50S subunit [262]. The switch loop, which connects domain 3 and 4 of a release factor, rearranges and interacts with universally conserved nucleotides that reside in the ribosomal A‐ site A1492 and A1493 in 16S rRNA, A1913 in 23S rRNA and protein S12 in the 30S subunit [248,

249]. This rearrangement is critical for correct positioning of the GGQ motif in the ribosomal peptidyl‐transferase center (PTC) of the 50S subunit and likely plays a role in signal transduction from the decoding center to the PTC [248, 249, 262]. In addition, the two neighboring glycines in the GGQ seem to adopt a backbone conformation needed for the movement of U2585, away from the ester bond of peptidyl tRNA [262] to expose it to

32 nucleophilic attack by water [263], and can explain the drastic reduction in RF activity upon their mutation [252, 255, 264].

RF1 is encoded by the prfA gene, and a mutant release factor 1, prfA1, has a clear temperature

–sensitive (Ts) phenotype at 42°C, increased misreading of both stop codons AUU and UAG, and enhanced efficiency for some tRNA nonsense suppressors [265, 266]. The mutant RF1 has an Arg to Pro change at position 137, within domain II, which probably leads to impaired binding of RF1 to the ribosome and as a consequence to reduced translation termination efficiency [267, 268]. There are many explanations for why a mutant allele of RF1 causes temperature sensitive growth at 42°C. It was shown for instance that the mutant RF1 terminates more slowly than the wild type at 37°C [268]. It is even possible that there is no termination at all at high temperature which results in ribosome stalling on mRNA with genes ending with stop codon UAG (M. Ryden‐Aulin, personal communication). Under this condition, one or few essential proteins will not express in the cell and the cell will either die or develop a suppressor mutation. One suppressor of the Ts‐phenotype has been isolated and a mechanism behind the suppression is discussed in paper III.

33 2. Results and discussion

2.1 Suppression of a cold‐sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region (paper I)

Translation initiation factor 1 (IF1) is an essential protein which has been found in all organisms [154, 155]. IF1 has been the subject of intensive research for many decades and several functions have been attributed to it. For instance, IF1 was suggested to interact with

RNA by having RNA chaperone activity [106, 172]. To find functional interaction that IF1 may have with rRNA and to learn more about IF1 function in the cell, we selected for second‐site suppressors of a defective cold‐sensitive IF1 mutant R69L of E.coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid, pKK3535, in a strain, JB69, with all chromosomal rRNA operons deleted. A set of suppressor mutations located in the processing stem of precursor 23S rRNA were isolated and the strains called (JB69/pD1, pD3 and pD6) depending on the location of the suppressor mutations in the plasmid. These mutations were found to interfere with processing of the 23S rRNA termini. That the suppressor mutations are not located in the structural part of the mature 23S rRNA suggests that the suppression mechanism is indirect and results from some rRNA maturation defect. This was supported by the observation that a lesion in RNase III, the enzyme involved in the initial cleavage of the processing stem, could suppress the IF1 defect and that a general defect in 50S subunit maturation could not suppress the cold sensitivity.

Analysing the sucrose gradient ribosomal profiles showed an increase in the relative amount of 70S particles as compared with free subunits in the IF1 mutant strain at nonpermissive temperature (23°C). Whereas a slight decrease in the extent of subunit association was found in the suppressor strains like (JB69/pD3). Our results suggest that the growth defect in the mutant

IF1 strain is at the level of ribosome subunit joining and that the suppressor mutations that specifically alter 23S rRNA processing partially restores the growth defect by limiting the available pool of mature 50S subunits. Moreover, a similarly large fraction of the 50S and the

70S ribosomes of one of the processing stem mutant (pD3) is composed of immature rRNA which means that there is no active system that degrades the immature 50S subunits or that

34 prevents them from entering the translating pool. However, a more realistic explanation can be that the assembly of ribosomal proteins onto the immature rRNA is delayed in these mutants, leading to an apparent decrease in the amount of ribosome subunits.

We found also that, in the case of the wild type plasmid pKK3535 irrespective of strain background, there was an increase in the relative amount of the fully mature terminus in the

70S fraction compared to the 50S fraction. The extended 23S rRNA termini were incorporated into the 50S subunits and the functional 70S ribosome. However the amount of the immature termini in the 70S ribosome was decreased compared to the fully mature termini suggesting that the final rRNA maturation occurs on translating ribosomes supporting the evidence that final maturation of the ribosome takes place during translation initiation [29].

It has been shown that IF1 stimulates the formation of the 30S initiation complex by inducing a conformation change over a long distance in the small ribosomal subunit [14, 163, 166, 269] and thus influencing the ribosomal subunit association‐dissociation rate [270], which is especially important in the cold [271]. IF1 is also shown to play a role in the cold shock response

[271‐274]. Moreover, recent data have suggested that IF1 is involved in subunit joining and has the ability to discriminate between certain mRNAs on the basis of their TIRs [174]. All these data and our results helped us to suggest a model for what happens in the IF1 mutant strain in cold based on the model proposed by Jonas et al [107]. Jonas et al suggested that cold shock conditions induce a conformation in the ribosome that becomes blocked in translation initiation, whereby only specific mRNAs with appropriate TIR can bypass this block and be translated.

We proposed that IF1 is involved in this mechanism by its ability to discriminate between certain mRNAs on the basis of their TIR. Depending on the TIR elements in mRNA, IF1 can either stabilize this conformation so that no translation occurs or induce a conformation change in the 30S subunit so that the rate of association‐dissociation with the 50S subunit is affected so that special mRNAs can be translated. The mutant IF1 fails to discriminate between certain TIR elements in mRNA and thus induces a conformation change in the 30S independently of this factor so that premature subunit joining is facilitated and translation of many unfavourable genes in the cold occur leading to cold sensitivity.

35 2.2 Influences of a mutated translation initiation factor IF1 or kasugamycin on Escherichia coli gene expression (Paper II)

Previous study has shown that IF1 cold‐sensitive mutation and kasugamycin addition lead to increased expression of reporter genes in a TIR dependent manner [234]. We have here investigated if a cold‐sensitive mutated IF1 and kasugamycin addition have the same effect on protein expression levels of natural genes using a 2D gel electrophoresis technique. Twenty‐one protein spots as a response to either the IF1 mutation or the addition of kasugamycin to the parental MG1655 strain were identified and quantified by mass spectrometry. Expression of several proteins was affected similarly under both conditions. However the effect was much more dramatic when the cell was treated with kasugamycin compared to the effect by mutant

IF1. While most of the proteins showed altered expression level under the influence of kasugamycin, more than a dozen showed no or only little changes in the case of IF1 mutant.

Moreover, in the case of kasugamycin treatment the changes in expression level were more than

3 times for a number of spots whereas most of the changes for the IF1 mutant strain did not exceed a factor of 2 times. Whereas the highest expression in the IF1 mutant strain was found for the YbgF protein (> 5 times) and in the relative abundance of the ribosomal protein S6 isoforms, the biggest influence on expression by kasugamycin were observed for the OppA protein and GroEL chaperonin protein. In addition, the expression of YbgF was also much increased after the treatment with kasugamycin as in the case of the IF1 mutant strain. The most prominent feature in the study is that gene ybgF seems to be uniquely sensitive to the IF1 mutation as well as upon addition of kasugamycin to the wild type strain. The YbgF protein is a part of the Tol‐Pal protein complex, which is involved in maintaining outer membrane integrity

[275, 276], but the function of YbgF is still unknown.

To gain more information about the similarity in the function between the altered IF1 mutation and the addition of kasugamycin, we chose to compare the effect between these two cases. To get a comparison between these two conditions, the response on expression obtained after the treatment with kasugamycin was divided with the one obtained when IF1 was mutated. Our results suggested that the effect on expression by kasugamycin was quite

36 different from that caused by a mutated IF1. Several genes were decreased in expression by one condition but increased in expression by the other. In addition, the effect in protein expression by kasugamycin, as compared to the IF1 mutant, was also much more drastic. While the expression of some genes by kasugamycin were relatively increased the expression of some other were dramatically decreased and this effect could be consistent with a pleiotropic mode of action by kasugamycin [277]. To explain the results, we suggested that kasugamycin may induce a special translation block for expression of certain genes in agreement with an earlier data showing that kasugamycin does not inhibit translation of all mRNAs equally well [216].

The high induction of GroEL chaperone complex by kasugamycin does not tell us to what extent the addition of kasugamycin can induce mistranslated and thus misfolded proteins. Since a similar induction of GroEL chaperone complex could not be found in cells treated with kanamycin known to induce mistranslated protein. Additionally, GroEL has been shown to facilitate the folding of no more than 5% of all proteins in the cell (reviewed in [278]).

As mentioned previously, several genes were affected similarly by an altered IF1 and by addition of kasugamycin. TIR regions of four genes with increased expression and four genes with decreased expression were cloned into the protein A’ reporter system [279] to investigate if the change in expression is a direct effect manifested at the level of translation initiation. TIRs representing several gene sequences gave similar increased expression both in the IF1 mutant and after the addition of kasugamycin. TIRs that gave high expression seem to have longer SD and longer AU‐rich regions. TIR sequence of the ybgF gave the highest increased expression under both conditions. This suggests that the elevated expression of the gene ybgF on the 2‐D gels is the result of a direct TIR influence at the level of translation initiation for this gene.

2.3 Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature‐sensitive phenotype caused by mutant release factor (paper III)

Bacterial release factor RF1 mediates termination of protein synthesis, specifically recognizing stop codons UAG and UAA [238‐240]. RF1 is encoded by the prfA gene, and a mutant release factor 1, prfA1, has a clear temperature –sensitive (Ts) phenotype at 42°C [265,

37 266]. The mutation affects the structure of RF1 central domain and probably leads to impaired binding of RF1 to the ribosome and as a consequence to reduced translation termination efficiency [267, 268].

We have mapped and characterized a suppressor of the temperature‐sensitive allele of RF1.

This suppressor mutation is linked to an IS10 insertion into the cysB gene, a transcription factor regulating the cys operon and leading to a Cys‐ phenotype. We have shown in the article that lack of CysB does not lead to increased expression of the prfA gene, and thus, the suppression phenotype is not a consequence of overexpression of the mutant RF1 protein. However, addition of high concentrations of cysteine to the growth medium restored temperature sensitive growth to the suppressed strain, and suggested that the suppression phenotype is caused by the lack of a component metabolized downstream of cysteine. Working on the hypothesis that deficiency of a thiolation reaction is responsible for suppression, we tested the involvement of several tRNA thiolation reactions. Our results showed that a temperature‐ sensitive mutation in ribosome release factor 1 is suppressed by mutations that result in loss of either 2‐thiolation or 5‐methylaminomethyl modification (mnm5s2U) of tRNA.

To address the question why a mutant allele of RF1 causes a temperature‐sensitive growth phenotype and to understand how the lack of the 5‐methylaminomethyl‐2‐thiouridine

(mnm5s2U) of the wobble base of tRNAGlu, tRNALys, and/or tRNAGln can suppress the Ts phenotype, we speculate that a mutant RF1, known to terminate slower at 37° C [268], fails to terminate at high temperature. In such a situation, the ribosome will stall at UAG stop codons and as a consequence one or more proteins, sometimes essential proteins, are not expressed and thus the cell will either die or develop a suppressor mutation to overcome the stalling. It is known that the mnm5s2U hypomodified tRNA can induce frameshifting, possibly caused by poor binding of the tRNA to the ribosome [280]. It has been also shown that the slippage in mutants defective in the synthesis of the modified nucleotide mnm5s2U increases when one of the codon for lysine or glutamine is placed in the P‐site, followed by a UAG stop codon [281].

Thus, if the gene that is not expressed due to stalling has a codon read by hypomodified tRNAs preceding the UAG stop codon, a frameshifting event would allow the ribosome to bypass the

38 stop codon in the +1 or ‐1 reading frame. This may permit ribosomes to terminate at an out of frame stop codon, UAA or UGA recognized by RF2, and a functional protein might be produced and hence suppression of the Ts phenotype.

3. Concluding remarks

IF1 has been the subject of intensive research for several years but the role of this protein in translation initiation remains unclear. We have now increased our knowledge about the IF1 function on the ribosome. We find that IF1 plays a role in ribosomal subunit joining and this function could be TIR‐dependent in cold. In the future, in vitro translation assay can help us to get more information about the suggested functions. This can be done by isolating the wild‐type and the mutant IF1, the 50S and the 30S subunits and the experiment can be run either in the cold or rooms temperature using different mRNA compositions designed previously [234].

We have also investigated if the IF1 mutant and the antibiotic kasugamycin affect the same related step in translation initiation. A 2D gel electrophoresis technique has helped us to gain an overview of proteins expressed under these two conditions. We find that the ybgF gene is uniquely sensitive to the IF1 mutation as well as to the addition of kasugamycin. The results have also suggested that the TIR region of ybgF may have some unique properties that influence expression of ygbF at the early translational phase. The unique property of the TIR region of the ybgF gene needs to be identified and investigated more closely. This can be done in several ways, one obvious way is to mutate systematically sequences in this region and see how this affects the expression of the gene using the protein A’expression assay system.

Finally, the results in this thesis give an important contribution for understanding translation termination. It supports the hypothesis from other researchers that modified nucleosides in the anticodons of tRNAs act in part to prevent frameshifting by the ribosome. We found that the mnm5s2U hypomodified tRNAGlu, tRNALys, and/or tRNAGln can suppress the Ts phenotype, caused by a mutant allele of RF1, by inducing frameshifting which leads to bypassing of the ribosome stalling at UAG stop codon in the + 1 or ‐1 reading frame. It will be interesting to see how the insertion in cysB does affect the levels of thionucleosides in the suppressed RF1 mutant strain′s tRNA and compare it with the effect caused by mnmA or tusB knockout.

39 4. Acknowledgements

I would like to thank

My supervisors, Prof. Leif Isaksson and Docent. Monica Rydén‐Aulin for accepting me as your

Ph.D. student and for all the time you spent on my articles. This thesis would not have been possible without your encouragement, guidance and support.

Jaroslav Belotserkovsky for good collaboration, for your endless help, for English proofreading this thesis and giving comments.

Sergey Surkov for nice collaboration, for sharing your experiences and helpful discussions.

Prof. Elisabeth Haggård for accepting me for a degree project, for your endless help and support and for critical reading of this thesis.

Dr. Anders Nilsson for your guidance during my degree project and for your friendship and help.

Prof. Ann‐Beth Jonsson for all your help during the dissertation process.

Dr. Margareta Ohné for your help during the lab courses.

Natalia Kotova for being a good friend at GMT.

Ingabritt Olausson and Görel Lindberg for perfect technical support and for your friendly attitude.

Eva Pettersson, Anette Storbacka and Eva Eyton for your administrative help.

All the present and former members at the GMT department for being pleasant colleagues during these years.

Finally, I am heartily thankful to all my family, especially my mother, my husband Riad who supported me during these years, and my children Nour and Rickard for making my life sunny.

40 5. References

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