studies on the functional interaction of translation initiation factor …516680/fulltext01.pdf ·...

Studies on the functional interaction of translation

initiation factor IF1 with ribosomal RNA

Jaroslav Belotserkovsky

Department of Genetics, Microbiology and Toxicology

Stockholm University, Sweden 2012

Interaction of IF1 with ribosomal RNA J. Belotserkovsky

© Jaroslav Belotserkovsky, Stockholm 2012

ISBN 978-91-7447-520-3 (pp. 1-44)

Printed in Sweden by Universtitetsservice US-AB, Stockholm 2012

Distributor: Department of Genetics, Microbiology and Toxicology

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Dedicated to my parents

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Abstract

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded

by a single infA gene in bacteria. Although several important functions have been

attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known

that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts

ribosomal RNA (rRNA) and induces large scale conformational changes in the small

ribosomal subunit. To shed more light on the function of IF1 and its interaction with the

ribosome, we have employed a genetic approach to elucidate structure-function

interactions between IF1 and rRNA. A selection has been used to isolate second site

suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with

an arginine to leucine substitution in position 69 (R69L). This yielded two classes of

suppressors – one class that mapped to the processing stem of 23S rRNA – a transient

structure important for proper maturation of 23S rRNA; and the other class to the

functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S

rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that

at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at

the level of ribosomal subunit association. These results led to a model whereby the cold

sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties,

while the 23S processing stem mutations indirectly suppress this effect by decreasing the

pool of mature 50S subunits available for association. Spontaneous suppressor mutations

in 16S rRNA were diverse in position and phenotypic properties, but all mutations

affected ribosomal subunit association, in most cases by directly decreasing the affinity of

the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA

yielded additional suppressor mutations that were localized to the mRNA and

streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA

suppressors occur in positions that affect the conformational dynamics brought about by

IF1. Taken together, this work indicates that the major function of IF1 is the modulation of

ribosomal subunit association brought about through conformational changes of the 30S

subunit.

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List of publications

This thesis is based on the following articles, referred to in the text by roman numerals:

I. Belotserkovsky JM, Isak GI, Isaksson LA. (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. Belotserkovsky JM, Dabbs ER, Isaksson LA. (2011). Mutations in 16S rRNA that

suppress cold-sensitive initiation factor 1 affect ribosomal subunit association.

FEBS J. 278(18):3508-17.

III. Belotserkovsky JM, Isaksson LA. (2012). Mutations in the streptomycin and

mRNA binding sites on 16S rRNA suppress a cold sensitive initiation factor

IF1. (Manuscript).

Papers I and II are reproduced with permission from the publishers.

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http://www.ncbi.nlm.nih.gov/pubmed/21418143





Abbreviations

rRNA ribosomal RNA

mRNA messenger RNA

tRNA transfer RNA

fMet-tRNA fMet formylated initiator tRNA

r-protein ribosomal protein

IF initiation factor

EF elongation factor

A-site aminoacyl

P-site peptidyl

E-site exit

h helix

DC decoding centre

PTC peptidyl transferase centre

GTP guanosine triphosphate

RRF ribosome release factor

SD Shine-Dalgarno

S Svedberg unit

antiSD anti-Shine-Dalgarno

Å angstrom

ASL anti-codon stem loop

TIR translation initiation region

OB oligomer binding

NMR nuclear magnetic resonance

IC initiation complex

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Table of contents

1. Introduction………………………………………………………………………..8

1.1 Structural components of the ribosome……………………………………......8

1.1.1 The small ribosomal subunit and 16S rRNA……………………....9

1.1.2 The large ribosomal subunit and 23S rRNA……………………...14

1.2 Transcription and maturation of ribosomal RNA…………………………….15

1.3 Translation initiation………………………………………………………….16

1.3.1 IF1 and its interaction with the ribosome…………………………20

2. Results and discussion…………………………………………………………….24

2.1 What is wrong with the R69L IF1 mutant? ......................................................24

2.2 Mode of action of 23S processing stem suppressors (Paper I)………………..27

2.3 Mode of action of 16S rRNA suppressors (Papers II and III)………………...29

3. Conclusions and future perspectives……………………………………………...31

4. Acknowledgments………………………………………………………………...32

5. References………………………………………………………………………...34

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1. Introduction

If catalysis of biological reactions by protein enzymes is central to the process of life, then

it must be acknowledged that the biogenesis of such proteins is paramount. The ribosome

is a molecular multi-component machine that is responsible for decoding the genetic code

in order to manufacture proteins. This process, termed translation, takes place in all living

cells on the planet using the same basic mechanism and components. This is so because

the apparatus of translation evolved early in the evolution of life, thus accounting for the

high degree of conservation in most of its components. The ribosome machine is

composed of both proteins (r-proteins) and RNA (rRNA). The ribosome itself is a catalyst

involved in the peptidyl transfer reaction, however, it is not the protein component, but the

rRNA that serves the primary catalytic function. Thus, the ribosome is in fact a ribozyme

(Ban et al., 2000; Nissen et al., 2000).

Generally, the process of bacterial translation as directed by the ribosome, occurs in four

stages – initiation, elongation, termination and recycling. During initiation, the ribosome

assembles on the mRNA with the aid of initiation factors (IFs) and initiator tRNA. The

ribosome then begins to ‘read’ the genetic code of the mRNA in three letter intervals (one

codon at a time) and translates the message into a polypeptide with the aid of elongation

factors (EFs) – this is termed elongation. When one of three stop codons is encountered,

the ribosome terminates translation with the aid of release factors (RFs). Finally, the

termination complex is recycled by release of tRNA and release factors accompanied by

the dissociation of the ribosome into the small and large subunits.

1.1 Structural components of the ribosome

In all organisms, the ribosome is composed of two interacting subunits that serve distinct

roles in translation – the small subunit, whose primary function is the decoding, and the

large subunit that is responsible for peptide bond formation. In prokaryotes, the small

subunit sediments at 30S and is approximately 0.8 megadaltons in mass; while the

corresponding values are 50S and 1.5 megadaltons for the large subunit. The translating

ribosome is comprised of a 1:1 stoichiometry of the small and the large subunits that

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sediments at 70S. While it has been known for some time that the ribosome is composed

of about two thirds RNA and one third protein, historically, it has been assumed that it is

the protein component that is responsible for the catalytic function. Early seminal work by

Dabbs was used to show that many of the proteins that comprise the 30S and 50S subunits

were in fact dispensable (Dabbs, 1978; Dabbs, 1979). With the discovery of catalytic RNA

– the ribozymes (Kruger et al., 1982), and later the high resolution crystal structures of

ribosomes (Ban et al., 2000; Nissen et al., 2000), it became evident that it is the RNA

component of the ribosome that carries out peptide bond catalysis and the decoding

functions (Moore & Steitz, 2011).

The ribosome contains 3 binding sites for tRNA – the A-site (aminoacyl), P-site (peptidyl)

and E-site (exit). These sites are structurally shared between the small and the large

subunits. In addition, there are tunnels for the passage of mRNA and the newly formed

polypeptide in the small and large subunits respectively. Besides these structurally defined

binding sites, there exist other overlapping and sometimes competing sites for initiation,

elongation and termination factors as well as other accessory proteins (Schmeing &

Ramakrishnan, 2009). These sites become available for binding at various stages of

ribosome function as viewed from a perspective of confirmation changes.

1.1.1 The small ribosomal subunit and 16S rRNA

The 30S subunit is composed of one rRNA molecule that sediments around 16S and is

1542 bases in length in Escherichia coli. In addition, there are 21 r-proteins. The small

subunit has been crystallized alone (Wimberly et al., 2000) and in complex with various

ligands such as initiation factors and antibiotics (Brodersen et al., 2000; Carter et al.,

2000; Carter et al., 2001). Based on these, and earlier studies, the small subunit has been

assigned structural features that generally correspond to domains in 16S rRNA (Yusupov

et al., 2001). The side of the 30S subunit that makes contacts to the 50S subunit is called

the interface side. This part of the 30S is almost entirely composed of rRNA, while the

solvent side is more r-protein dominated. In addition, most of the contact points between

the 30S and 50S subunit – the bridges, are made up of rRNA. There are some 12 bridges

between the subunits, mostly made up of rRNA contacts (Yusupov et al., 2001). The

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importance of inter-subunit bridges in ribosome function have been highlighted by

mutagenesis studies (Liiv & O'Connor, 2006; Sun et al., 2010). Helix 44 in 16S rRNA

houses 4 bridges (B2a, B3, B5 and B6) (Yusupov et al., 2001). This helix, comprising

most of the 3′ minor domain of 16S rRNA, is a prominent structural feature on the

interface side of the 30S subunit. Bridge B3 in h44 forms a pivoting point for the

ratcheting motion of the small subunit around the large subunit during translocation

(Dunkle et al., 2011; Jin et al., 2011).

Figure 1. The small ribosomal subunit from T. thermophilus. Views A and B are from the

interface and solvent sides respectively. rRNA is in grey, r-proteins are in color. Morphological

features are labeled appropriately. Adapted with permission from (Brodersen et al., 2002).

Copyright © 2002, Elsevier.

In addition to its contribution to inter-subunit bridges, h44 is the major player in the

decoding function of the small subunit due to its structural contribution to the A-site

(Moazed & Noller, 1986; Moazed & Noller, 1990; Ogle et al., 2001). During decoding,

the universally conserved bases A1492 and A1493 in h44 interact with the minor groove

formed by the three base pair mini-helix of the cognate mRNA codon and tRNA

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anticodon. The adenine of A1493 makes A-minor interactions with the first base pair of

the codon – anticodon while A1492 together with G530 monitor the second position. The

presence of the cognate anti-codon triggers a displacement of A1492 and A1493 from h44

and a rotation of G530 from the syn to the anti conformation (Ogle et al., 2001). These

local sensors of the cognate geometry of codon-anticodon in the decoding center (DC)

trigger large scale conformational changes in the 30S subunit (domain closure), with most

prominent movements in the head and the shoulder domains of the subunit (Ogle et al.,

2002). While not entirely understood, these movements most likely contribute to the

hydrolysis of GTP by EF-Tu during the proof-reading phase of decoding (Jenner et al.,

2010a; Moore & Steitz, 2011; Schmeing et al., 2009). This model - whereby G530, A1492

and A1493 are active players in decoding, has recently been challenged (Demeshkina et

al., 2012). Those authors present evidence that these 16S rRNA bases do not actively

sense the cognate codon-anticodon geometry. Instead, it appears that both cognate and

near-cognate tRNAs in the A-site are able to induce domain closure with equivalent

displacements of G530, A1492 and A1493 - making these bases a static feature of the A-

site. The discrimination against near-cognate tRNAs is brought about by the strict

structural constraints on the codon-anticodon mini-helix. This is because the first two

bases of the codon are limited to form Watson-Crick base pairs. Presumably this will not

be the last word on the mechanism of decoding by the ribosome, it is clear however, that

the universally conserved bases G530, A1492 and A1493 are central in this process.

Helix 18 which houses the 530 loop, and in turn the aforementioned universally conserved

G530 (Cannone et al., 2002), forms part of the shoulder of 30S. It is sandwiched between

proteins S4 on the solvent side, and S12 on the interface side – which connects it to h44.

In addition, h18 lies in close proximity to the central pseudoknot made up of helices 1 and

2 that serves as a central juncture between the 5′, 3′ and central domains of 16S

(Brodersen et al., 2002). Bases 505-507 and 524-526 in h18 participate in a pseudoknot

structure whose importance has been highlighted by mutagenic studies (Powers & Noller,

1991). The 530 loop, together with r-protein S12 and bases in helices 44, and 34 in the

head of the subunit, form the A-site (Moazed & Noller, 1986; Moazed & Noller, 1990;

Ogle et al., 2001).

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Contacting S12, and the central pseudoknot is h27 which forms part of the central domain

of 16S rRNA. Functionally important bases in this helix include C912, mutations in which

give rise to streptomycin resistance (Frattali et al., 1990; Gregory & Dahlberg, 2009;

Montandon et al., 1986), and the 900 tetraloop, that participates in the formation of bridge

B2c (Yusupov et al., 2001). The central domain of 16S rRNA makes up most of the

platform of the small subunit (Brodersen et al., 2002). The juncture between helices 20, 21

and 22 is the binding site for the primary binding protein S15, that in turn participates in

the formation of bridge B4 (Brodersen et al., 2002; Serganov et al., 1996; Yusupov et al.,

2001). Functionally important rRNA bases in the central domain include residues in the

790 region (h24), G926 and G966, that together with bases in the 3′ major domain –

A1339 and G1338, and 3′ minor domain bases C1400, C1402 and U1498 structurally

contribute to the ribosomal P-site. The 790 loop and A1339 and G1338 form a gate that

physically separates the P and E sites on the small subunit. Importantly, A1339 and G1338

interact with the anticodon arm of P-site bound fMet-tRNAfMet through a type I and type II

A-minor interaction, contributing to discrimination of initiator tRNA during initiation

(Berk et al., 2006; Korostelev et al., 2006; Lancaster & Noller, 2005; Moazed & Noller,

1990; Selmer et al., 2006).

The 3′ major domain of 16S rRNA makes up the head of the small subunit (Brodersen et

al., 2002). Significant dynamic movements during initiation, elongation and termination

occur in the head domain of the small subunit (Berk et al., 2006; Carter et al., 2001; Julian

et al., 2011; Korostelev et al., 2008; Schuwirth et al., 2005). Ribosomal proteins S9 and

S13 in the head of the subunit have C terminal extensions that also contribute to the P-site

(on the boundary between A and P-sites) and may be major players as signaling conduits

from the decoding centre (DC) to the peptidyl-transferase center (PTC) during the

proofreading step (Jenner et al., 2010a; Jin et al., 2011). Partial or complete deletion

mutants of S9 and / or S13 are viable but have significant defects associated with growth,

subunit association, translocation and tRNA binding (Cukras & Green, 2005; Hoang et al.,

2004).

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Figure 2. The path of mRNA on the ribosome. Panel A depicts the path of the mRNA on the

30S subunit. The features of mRNA are: Shine-Dalgarno in bright yellow; E-site codon in blue; P-

site codon in red; A-site codon in orange and downstream region in dark yellow. Morphological

features of the small subunit are labeled. Note that ‘beak’ of the small subunit is misspelled. Panel

B shows the detail of the downstream interaction of mRNA bases with 16S rRNA bases in helix

34 – a stacking network between positions +8 and +9 in mRNA with C1054 and U1196. Adapted

with permission from (Demeshkina et al., 2010). Copyright © 2010, Elsevier.

Helix 34 comprises the lower part of the head of the subunit and is adjacent to the

shoulder domain. Bases G1207 of h34 and U531 of h18 interact via a sugar-sugar H-bond.

Mutagenic (Jemiolo et al., 1991; Pagel et al., 1997) and biochemical (Dontsova et al.,

1992; Matassova et al., 2001) data indicate that h34, and in particular base C1054 plays a

functionally important role in the A-site – according to structural data, packing against the

wobble mRNA-tRNA base pair (Jenner et al., 2010b; Selmer et al., 2006). S3 interacts

with h34 on the solvent side of the subunit which together with S4 and S5 form part of the

downstream tunnel for the passage of mRNA through the ribosome (Jenner et al., 2010b;

Takyar et al., 2005; Yusupova et al., 2001). On the opposite side, around the neck of the

subunit, is the upstream mRNA binding site. This site is comprised of helices 20, 28 and

37 in rRNA, as well as proteins S7, S11 and S18 (Yusupova et al., 2001). S7 seems to be

the major contact point for interaction with E-site tRNA on the small subunit (Korostelev

et al., 2006; Selmer et al., 2006), explaining the lack of protections of 16S rRNA from

chemical probes by E-site tRNA (Moazed & Noller, 1986; Moazed & Noller, 1990).

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1.1.2 The large ribosomal subunit and 23S rRNA

The large ribosomal subunit is composed of two RNA molecules – the 2900 bases long

23S and the 120 bases long 5S rRNA, as well as 34 r-proteins. The general architecture of

the 50S subunit is more monolithic than 30S – being of a globular nature with three

protuberances (Ban et al., 2000). These are the L7/L12 stalk, the central protuberance

(CP) – composed primarily of 5S rRNA, and the L1 stalk (Mueller et al., 2000; Penczek et

al., 1999). There are six 23S rRNA domains, with the seventh made up of 5S rRNA. In

contrast to the small subunit and 16S rRNA domain correspondence, the folding of the

domains of 23S rRNA gives rise to a more compact intertwined structure (Harms et al.,

2001). As with the small subunit, the active site of the large subunit is composed of rRNA

(Nissen et al., 2000). In addition, although the ribosome is asymmetric, the rRNA helices

that make up the A and P sites on the large subunit form a pseudo-symmetrical structure,

representing an evolutionary vestige of a proto-ribosome (Bashan et al., 2003; Krupkin et

al., 2011).

The catalytic site – peptidyl-transferase centre (PTC), which accommodates the CCA ends

of A-site and P-site tRNAs, is located in domain five (V) of 23s rRNA (Nissen et al.,

2000). Structural evidence indicates that N3 of A2451 and the 2′OH group of A76 of the

P-site tRNA are in close proximity to the α-amino group of the attacking A-site tRNA, and

only these could play a direct role in peptide bond catalysis in the PTC (Nissen et al.,

2000). Extensive mutagenic analysis has revealed that A2451 is essential for peptide

hydrolysis during termination, but not for peptide bond formation (Youngman et al.,

2004). Thus, its contribution to direct catalysis has been all but ruled out. However, more

recent studies show that that A2541 (E. coli) plays a more active role than previously

concluded (Lang et al., 2008). In contrast, evidence suggests that the 2′OH group of A76

of P-site tRNA plays an essential role in peptide bond catalysis (Dorner et al., 2003;

Weinger et al., 2004). In general, it is believed that the contribution of the PTC to peptide

bond formation is largely due to substrate positioning (Nierhaus, 1980; Sievers et al.,

2004), with other mechanisms, such as transition state stabilization possibly also playing a

role (Hiller et al., 2011). Although rRNA is the main component of the PTC, more recent

crystal structures indicate that the N-terminus of L27 interacts with the tRNA substrates

(Voorhees et al., 2009). Finally, it is of note that the PTC prevents premature hydrolysis of

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the P-site substrate by an induced fit mechanism, whereby binding of the amino-acyl

tRNA induces specific movements of the PTC positioning the P-site substrate for attack

(Schmeing et al., 2005).

1.2 Transcription and maturation of ribosomal RNA

Typically, there are multiple rRNA operons in most organisms. E. coli has 7 rRNA

operons including rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH and apart from a few

sequence differences, the rRNA genes are similarly arranged in the order – 16S, 23S and

5S. In addition, there are either one or two tRNA genes in the spacer region between 16S

and 23S genes as well as tRNA genes downstream of the 5S gene. The rRNA operons are

transcribed as one primary transcript under the control of rrn P1 and rrn P2 promoters,

where the former is stronger and of more relevance during fast growth, while the latter

accounts for the majority of transcription during slow growth. The P1 promoter is

preceded by AT rich upstream (UP) elements as well as number of binding sites for the

transcription factor Fis. Both these features enhance the binding of RNA polymerase

through interaction with its C-terminal domain of the α subunits, thus accounting for the

increased activity of P1 during fast growth (for extensive reviews see (Condon et al.,

1995; Paul et al., 2004)).

The process of rRNA maturation and folding begins as soon as rRNA emerges from the

RNA polymerase. As transcription proceeds, there is formation of secondary structures

within rRNA sequences that occur spontaneously, or are stimulated by the binding of

ribosomal proteins. Thus, the 5′ to 3′ directionality of transcription sets the order of

binding of r-proteins (reviewed in (Woodson, 2008)). However, interestingly, it has been

demonstrated that the order of transcription of domains within either 16S or 23S rRNA

genes is not critical for proper ribosome maturation and function. This, because circular

permutations of 16S or 23S rRNA transcripts were functional and supported the growth of

a cell as sole sources of rRNA to near wild type extent (Kitahara & Suzuki, 2009). During

the later stages of transcription, transient complexes within the primary transcript form

and aid in the correct folding of rRNA domains and assembly of the subunits (Liiv &

Remme, 1998; Liiv & Remme, 2004). The importance of these transient interactions was

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highlighted by the observation that mutations within the leader sequence preceding 16S

rRNA give rise to aberrant 30S particles, even though the leader sequence does not form

the structural part of mature 16S rRNA (Schaferkordt & Wagner, 2001; Theissen et al.,

1993). Such transient structures are eventually outcompeted by more stable stem structures

(processing stems) that are comprised of the terminal sequences of 16S, 23S and 5S genes

(Young & Steitz, 1978). These are substrates for RNase III which initiates a chain of

nucleolytic processing events eventually leading to the formation of mature 16S and 23S

rRNA. The maturation of 16S rRNA is not strictly dependent on the activity of RNase III,

with maturation of the 5′ end a function of RNase E and RNase G, and 3′ processed by an

as yet unidentified ribonuclease. In contrast, processing of 23S rRNA is strictly dependent

on the action of RNase III. Strains with a deletion in rnc – a gene encoding RNase III, are

viable but have a heterogeneous population of immature termini that never mature (King

et al., 1984) (thoroughly reviewed in (Deutscher, 2009). Proper formation of the

processing stem is required for efficient maturation of 23S rRNA since mutations that

interfere or abolish the formation of this structure interfere with 23S maturation

(Belotserkovsky et al., 2011b; Liiv & Remme, 1998).

1.3 Translation initiation

The initiation phase of translation can be regarded as the most important phase with regard

to gene expression. Here, mRNA binds to the 30S subunit with the participation of the

initiator fMet-tRNAfMet and initiation factors IF1, IF2 and IF3 all forming the 30S pre-

initiation complex (Gualerzi et al., 2001). The basic function is to select the correct start

codon on the mRNA in order to proceed with elongation. This is achieved by a codon-

anticodon interaction of mRNA with fMet-tRNAfMet respectively as directed by trans-

acting initiation factors (Laursen et al., 2005; Simonetti et al., 2009).

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Figure 3. Schematic of the steps in the initiation of translation. The small and large ribosomal

subunits are in light grey and dark grey respectively. Individual components are labelled. Adapted

with permission from (Laursen et al., 2005). Copyright © 2005, American Society for

Microbiology.

Figure 3 shows the basic outline of the steps involved in translation initiation. To

commence initiation, the ribosomal subunits need to be dissociated following the

termination phase of translation in order to supply new subunits for initiation (Janosi et

al., 1996; Kisselev et al., 2003). This is achieved by the action of ribosome recycling

factor (RRF) and EF-G on post termination ribosomes containing P-site tRNA and

mRNA, or as a complementary pathway, by IF1 and IF3 on ribosomes with mRNA and an

empty P-site (Pavlov et al., 2008). Following splitting of 70S into subunits, IF3 remains

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bound to free 30S particles to prevent re-association. Thus, IF3 serves an anti-association

function (Grunberg-Manago et al., 1975). This is followed by the binding of mRNA,

fMet-tRNAfMet, and IF2. The precise order and timing of these events is not known,

however a recent study indicates that the binding of IF2 precedes that, and stimulates the

binding of fMet-tRNAfMet to the 30S subunit (Milon et al., 2010). The binding of mRNA

occurs in several distinct steps. Initial binding – also called anchoring, occurs when

mRNA binds to the platform of 30S. Here, mRNA can be bound in a structured state, and

may or may not involve SD-antiSD interaction. This initial binding is mediated by r-

proteins S2, S7, S11 and S18 (Marzi et al., 2007; Simonetti et al., 2009). At this point, an

initiation milestone termed the ‘pre-initiation complex’, the anti-codon stem of fMet-

tRNAfMet is not properly positioned in the P-site, and the mRNA codon is in the ‘stand-by’

state (Kaminishi et al., 2007; La Teana et al., 1995). This step is followed by mRNA

‘accommodation’ into the mRNA channel around the neck of 30S with a defined SD-

antiSD interaction (Kaminishi et al., 2007; Simonetti et al., 2009; Yusupova et al., 2006).

The concerted action of IFs drives a conformational change upon which a codon-

anticodon interaction takes place in the P-site, resulting in a 30S initiation complex (30S

IC) formation - representing a state competent for association with the 50S subunit

(Gualerzi et al., 2001). In general, the process of initiation is a highly orchestrated and

inter-dependent set of events that requires all the above mentioned components for proper

function.

Major insights into specific steps in translation initiation have come from more recent

cryo-EM structures as well as rapid kinetic studies. Although of limited resolution,

structures of 70S ICs unambiguously place IF2 in the subunit cleft on the ribosome, the so

called ‘factor binding site’ shared by EF-G, EF-Tu and RF3 (Allen et al., 2005;

Myasnikov et al., 2005). On the 30S side, the N-terminal domain (NTD) of IF2 contacts

helices 5 and 14 in 16S rRNA, while the C terminal end contacts the conserved 3′ hexa-

nucleotide CAACCA of initiator tRNA (Julian et al., 2011; Simonetti et al., 2008), in

agreement with other studies (Guenneugues et al., 2000). Contacts to IF1 and S12 are also

made via the NTD of IF2, however, this is only evident in the E. coli structure (Julian et

al., 2011). In contrast, the T. thermophilus structure shows no such IF1-IF2 contact due to

a shorter NTD of T. thermophilus IF2 (Simonetti et al., 2008). This further supports the

notion that a physical IF1-IF2 interaction is not universally conserved and may not be

18


necessary (Julian et al., 2011; Kapralou et al., 2008). In agreement with earlier

suggestions, different complexes of 30S and 70S ICs show a series of conformational

states of IF2 in relation to the subunits, implying that IF2 assumes several distinct

conformations at different stages in initiation (Marzi et al., 2003; Myasnikov et al., 2005;

Simonetti et al., 2008; Simonetti et al., 2009).

Figure 4. Cryo-EM structure of the complete 30S initiation complex with IF1, IF2 and IF3 as

well as initiator tRNA. Two views of the complex from the shoulder and interface sides of the

small subunit. Atomic resolution structures are fitted onto the cryo-EM co-ordinates in the bottom.

Features of the small subunit are labelled: h is head; sh is shoulder; sp is spur; IF1 is in blue, IF2 is

in green; IF3 is beige; initiator tRNA is in orange. Adapted from (Julian et al., 2011). Copyright ©

2011, Julian et al. (open access).

An important finding from the 70S IC is that IF2 induces a rotation of the 30S relative 50S

following GTP hydrolysis (Myasnikov et al., 2005). This hydrolysis event returns the

ribosome to the classical ‘non-rotated’ state competent for elongation as further

corroborated by kinetic studies (Marshall et al., 2009). The binding of IF2 also induces a

rotation of the head of 30S (Julian et al., 2011). Another important observation from

structural data concerns the role of IF2 in stimulating subunit association. The

dimerization interface of the 30S and 50S subunits is approximately 6000 Å2 and is

19


composed almost exclusively of negatively charged rRNA. Thus there is a significant

electrostatic barrier that must be overcome to allow subunit joining. This explains the

requirement for Mg2+ ions for the formation of rRNA bridges. IF2, together with the other

IFs and Mg2+ lowers the electrostatic barrier by burying nearly 2600 Å2 of the dimerization

surface, thus favouring subunit association (Allen et al., 2005; Marshall et al., 2009)

Regarding the positioning of fMet-tRNAfMet , the visualized complexes of both the 70S IC

and the 30S IC indicate that the anticodon-stem-loop (ASL) is essentially in the P-site

while the acceptor stem is able to rotate between the P/I state – shifted towards the E-site

(Allen et al., 2005; Simonetti et al., 2008), or a novel P/I1 state – shifted towards the A-

site (Julian et al., 2011). This movement is mediated by contact with IF2 and may have a

functional relevance (Julian et al., 2011; Simonetti et al., 2009). The latest cryo-EM

structure also visualizes IF3 in the 30S IC (Julian et al., 2011). In the current structure, the

N-terminal domain contacts the elbow region of fMet-tRNAfMet while the C-terminal

domain contacts the 790 loop in h24 of 16S rRNA in agreement with earlier biochemical

evidence (Dallas & Noller, 2001). The position of IF3 in the 30S IC would block the

formation of bridge B2b, thereby preventing subunit joining and rationalizing its role as an

anti-association factor (Julian et al., 2011). However, comparing this 30S IC position of

IF3 and that of a modelled 70S IC suggests a conformational change of IF3 upon the

formation of the 70S IC (Allen et al., 2005; Julian et al., 2011). This conformational

change to allow formation of B2b is presumably related to the translation initiation region

(TIR) dependant discriminatory effect of IF3 observed in recent kinetic studies (Milon et

al., 2008).

1.3.1 IF1 and its interaction with the ribosome

Initiation factor 1 (IF1) is the smallest of the three factors involved in translation initiation

in E. coli (Sands et al., 1987). IF1 is transcribed as a monocistronic mRNA from the infA

gene with transcription initiated from two promoters – P1 and P2, and terminated by a ρ-

independent terminator (Cummings et al., 1991). The P2 promoter is the more active

promoter during normal growth, and is under metabolic control (the amount of IFs in the

cell increases with growth rate), while transcription from P1 is primarily induced under

20


cold shock (Giangrossi et al., 2007; Ko et al., 2006). IF1 does not auto-regulate

transcription or translation of infA (Cummings et al., 1991). Unlike IF3, IF1 (and IF2) is

conserved and its homologues are present in all three domains of life (Kyrpides & Woese,

1998). Interestingly, while mammalian mitochondria lack an IF1 homologue, it has been

shown that the mitochondrial IF2 (IF2mt) has an insertion domain that fulfils the

equivalent role of IF1 (Gaur et al., 2008; Yassin et al., 2011). IF1 is essential for cell

survival (Cummings & Hershey, 1994). This property has been exploited as a mechanism

to maintain plasmid stability without the use of antibiotic selection (Hagg et al., 2004).

The structure of bacterial IF1 has been solved both in solution (Sette et al., 1997) and in

complex with the 30S subunit (Carter et al., 2001). The former showed that IF1 belongs to

the oligomer binding (OB) family of proteins, members of which, such as CspA (cold

shock protein A) bind single and double stranded nucleic acids (Sette et al., 1997). Indeed

early studies have shown that IF1 can bind polynucleotides (Schleich et al., 1980). The

latter revealed that IF1 interacts with the 30S subunit in a cleft formed between r-protein

S12, helix 44 and the 530 loop of 16S rRNA, placing it in the A-site. Equivalently the

binding site of the eukaryotic homologue eIF1A (OB domain) has also been mapped to the

A-site (Yu et al., 2009). This justified previous suggestions that IF1 blocks the A-site from

elongator tRNA during initiation. Both structural (Carter et al., 2001) and biochemical

data (Moazed et al., 1995) indicate that IF1 makes contacts with the universally conserved

bases G530, A1492 and A1493 in 16S rRNA (Figure 5). These bases were further

implicated by mutational analysis as important sites for IF1 interaction (Dahlquist &

Puglisi, 2000). The functionally important residues Arg41 and Arg46 (40 and 45

respectively in E. coli) stack against A1492 and A1493, flipping them out. In addition,

there is a significant phosphate backbone interaction with the 530 loop. Local interactions

of IF1 with A-site components triggers a large scale conformational rearrangement, with a

prominent movement of the head of 30S. More specifically, the binding of IF1 results in a

disruption of base pairing of A1413-G1487 through interaction with the phosphate

backbone of h44 as assisted by S12. These bases, together with h44 base A1408 were

previously found to be important in IF1 binding, and more recently in IF1-dependent start

codon discrimination (Dahlquist & Puglisi, 2000; Qin et al., 2012). Unfortunately the

aforementioned T. thermophilus structure of IF1 in complex with 30S only contains a

small piece of polyU mRNA that does not extend significantly to reach the A-site.

However, the authors note that the binding of IF1 would contact but not block the mRNA

21


tunnel (Carter et al., 2001). In the downstream position of the A-site, IF1 is within

approximately 5Å of the universally conserved C1054 that monitors the third position of

the codon-anticodon (Jenner et al., 2010b; Selmer et al., 2006). In general, IF1 binds in a

1:1 stoichiometry to the 30S subunits, with electrostatic (ionic) interactions accounting for

most of the binding (Celano et al., 1988; Zucker & Hershey, 1986). In addition, IF1 has

very low affinity to the 50S subunit and does not form a stable interaction with the 70S

ribosome (Celano et al., 1988).

Figure 5. Structure of IF1 on the small ribosomal subunit from T. thermophilus. Panel A

shows the placement of IF1 in the A-site of the small subunit. IF1 is in green; rRNA is in grey; r-

proteins are in blue; a small piece of mRNA is in magenta; the position of R69 (R70 in T.

thermophilus) is in red. Panel B shows the detailed view of IF1 in the A-site. The individual

components are labelled; R69 is in red. Figure generated using PyMOL from PDB entry 1HR0

from (Carter et al., 2001).

Functionally important residues in E. coli IF1 were identified by NMR spectroscopy as

well as mutagenic studies (Croitoru et al., 2004; Gualerzi et al., 1989; Paci et al., 1983;

Sette et al., 1997; Spurio et al., 1991). Results from early NMR studies indicated that

some His and Arg residues were of importance for IF1 binding (Paci et al., 1983). Later

mutagenic data supported these observations and highlighted the role of His29 and His34

22


in ribosome binding and IF1 recycling. Of special importance was the C-terminal Arg69,

mutations in which resulted in severe binding defects as well as reduced activity in

promoting IC formation (Gualerzi et al., 1989; Spurio et al., 1991). This residue was also

singled out in mutagenic studies by Croitoru et al., who also systematically mutated all six

Arg residues (22, 40, 45, 65, 69 and 71) to Leu or Asp. It was found that mutations of

positively charged arginine residues to either neutral leucine or negatively charged

aspartic acid, gave rise to cold sensitivity in mutants, with the most prominent being R40D

and R69L (the corresponding R69D was not viable) (Croitoru et al., 2004). It was also

demonstrated that the R69L alteration gives rise to a general increase in test gene

expression compared to wild type, and this effect was especially relevant at reduced

temperature (Croitoru et al., 2005). Based on structural data, Arg69 (Arg70 in T.

thermophilus) is in position to interact with mRNA in the A-site (Carter et al., 2001),

although during the aforementioned binding experiments, mRNA was not included,

suggesting that Arg69 functionality (or loss of function in the case of a mutant) does not

strictly depend on mRNA interaction (Gualerzi et al., 1989). To further investigate the

interesting properties of this mutant, this work describes the selection of second site

suppressors in rRNA that rescue the cold sensitivity of R69L mutant of IF1

(Belotserkovsky et al., 2011a; Belotserkovsky et al., 2011b).

Although the precise function of IF1 remains elusive, this small factor has a number of

described effects in translation initiation as well as transcription. Importantly, IF1 plays a

role in the selection of fMet-tRNAfMet by stimulating the affinity enhancing action of IF2,

and the affinity reducing action of IF3 (Antoun et al., 2006a; Antoun et al., 2006b; Hartz

et al., 1989). IF1, together with IF3, prevents the formation of 70S IC that lack fMet-

tRNAfMet (Antoun et al., 2006a; Pavlov et al., 2008). IF1 together with IF2 aids in the

binding and unfolding of mRNA, most likely as a result of the stabilization of fMet-

tRNAfMet on the ribosome by the factors (Studer & Joseph, 2006). In general, IF1

stimulates the formation of the 30S IC (Pon & Gualerzi, 1984). Early studies indicate that

IF1 stimulates the association / dissociation rate of ribosomal subunits, without affecting

the equilibrium point (Grunberg-Manago et al., 1975; van der Hofstad et al., 1978).

However, more recent kinetic studies indicate that IF1 does indeed affect the equilibrium

point, possibly by stabilizing a certain conformation of 30S that has a high affinity for IF3

(Pavlov et al., 2008). Likely related to this, IF1 and IF3 are involved in an additional

23


proof-reading step at some point during 70S IC formation that discriminates against

mRNAs with certain non-favourable TIRs (Grigoriadou et al., 2007; Milon et al., 2008).

IF1 is a cold shock response protein – in that its transcription and translation are

stimulated by cold shock (Giangrossi et al., 2007; Ko et al., 2006). In addition, IF1 has

both RNA chaperone activity (Croitoru et al., 2006) and transcription anti-termination

activity (Phadtare et al., 2007), although the latter function is not essential in vivo. The

eukaryotic counterpart eIF1A also possesses RNA chaperone activity, and this property is

localized to the OB-fold domain of the protein (Kwon et al., 2007). Micro-array studies

show that a number of genes are responsive, at the transcription level, to over-expression

of IF1 (Phadtare & Severinov, 2009).

2. Results and discussion

2.1 What is wrong with the R69L IF1 mutant?

Of the nine chromosomal IF1 mutants constructed by Croitoru et al., the R40D and R69L

mutants displayed the most severe cold sensitive phenotype. The defect resulting form the

R40D alteration could be rationalized in view of the fact that this residue contacts the

universally conserved A1492 (Carter et al., 2001; Moazed et al., 1995). An improper

interaction due to this mutation could therefore disrupt the flipping out of A1492 observed

in the crystal structure, with associated downstream effects. On the other hand, the reason

behind the cold sensitivity of R69L mutant is not entirely clear, in part due to the absence

of full length mRNA in the current IF1-30S complex structure as mentioned above.

However, as has been suggested, the very cold sensitivity of these mutants can be

exploited as a means of selection for second site suppressor mutations in components that

interact with IF1 (Croitoru et al., 2004). This approach has been applied to study

functional interactions between IF1 and rRNA in papers I and II.

During our work with the R69L IF1 mutant, it became obvious that at least one of the

defects associated with this mutation was at the level of ribosomal subunit association, as

detected using the sucrose gradient experiments (Belotserkovsky et al., 2011a;

Belotserkovsky et al., 2011b). This defect was found specifically at the temperature

downshift condition (in the cold), and not 37°C. In the typical profile of the R69L IF1

24


mutant (either the MG1655 derived or SQZ10 derived) following the downshift, there is

an apparent relative decrease in the peak corresponding to the 50S subunit. Although less

obvious, there also seems to be an increase in the peak corresponding to the 70S

ribosomes (see Figures 5 and 7 in Paper I). There are at least two possible ways to explain

this observation:

1) There is a stoichiometric imbalance in the ribosomal subunits such that 30S

subunits are in access in the IF1 mutant in the cold. Although quantification of

molar ratios of subunits in Paper I does not support this explanation, it may still be

a formal possibility due to a limitation of the quantification method as discussed in

the paper. According to this view, such an imbalance may arise either due to the

overrepresentation of 30S subunits, or specific decrease in 50S subunits in the

mutant strain. In the case of the former, there may be an apparent accumulation of

30S subunits that are incompetent to associate with the 50S subunits due to the

defective action of mutant IF1. This may in turn lead to degradation of available

free 50S subunits, accounting for the stoichiometric imbalance. This possibility

can not be regarded since loss of free 50S subunits would lead to decreased 70S

formation – contrary to what is actually observed. In the case of the latter, specific

decrease in the 50S subunits due to the action of IF1 may be achieved due to the

defective anti-termination activity of IF1 on the rRNA transcript. As it has been

demonstrated that IF1 is an anti-terminator (Phadtare et al., 2007), this is indeed a

possibility, and stoichiometric imbalances of 30S to 50S subunits have been

reported with defects in NusA and NusB anti-terminators (Quan et al., 2005). We

have investigated this possibility with the use of radio-labelled probes for 16S, and

5S rRNA in Northern blotting experiments, as well as total RNA electrophoresis.

Comparing the IF1 mutant strain CVR69L to MG1655 when shifted to the cold,

similar ratios of 16S to 5S rRNA were observed (unpublished data not shown).

Thus, this possibility was rejected.

2) The stoichiometries of 30S and 50S subunits are unaltered, but there is increased

70S formation in the IF1 mutant in the cold. This view is supported by

experimental evidence in Paper I. It is known that IF1 plays a role in both the

formation of the 30S IC as well as assisting IF3 in maintaining a pool of free

25


subunits in the recycling step (Antoun et al., 2006b; Grunberg-Manago et al.,

1975; Pavlov et al., 2008). Thus, the defective action of IF1 accounting for

increased 70S formation may be either in the subunit association step (forward

reaction) or the 70S ribosome dissociation step (reverse reaction). To further

investigate this question, following the publication of Paper I and II, we used a

complementary sucrose gradient method that also resolves polysomes

(polyribosomes). Comparing the polysomes profiles of the IF1 mutant strain

CVR69L and its isogenic parental strain MG1655 indicates that the IF1 mutant has

decreased polysomes and free 30S and 50S subunits compared to wild type, with a

concurrent increase in the 70S ribosomes (Figure 6 in thesis, and Figure 2B in

Paper III). This effect is only observed upon downshift to the cold and not at 37°C.

Strikingly, an almost identical polysomes profile has been previously reported in

IF1 depletion experiments (Cummings & Hershey, 1994). Those authors

interpreted the results as decreased 70S IC formation due to the depletion

(inactivity) of IF1. However, this result would also be consistent with the role of

IF1 in the recycling step since the defective action of mutant (or depleted) IF1

could also affect the dissociation of post-termination 70S ribosomes into subunits

(Pavlov et al., 2008). In fact, it has been shown that an increasing relative amount

of IF1 (and IF3) is necessary in the cold in order to maintain an adequate supply of

free 30S subunits since 70S monosomes are more stable at reduced temperature.

This stoichiometric increase of the IF1 is due to the cold-shock response

(Giangrossi et al., 2007). Interestingly and somewhat counter-intuitively (since IF2

stimulates subunit association), when IF2 is depleted from the cell, there is a

similar increase in 70S monosomes and a decrease in polysomes (Cole et al.,

1987). A similar effect is also observed when cells are downshifted from rich to

poor media, and this is due to the accumulation of 70S ICs bound to mRNA but

blocked in translation (Jacobson & Baldassare, 1976; Ruscetti & Jacobson, 1972).

It has been demonstrated previously that the R69L IF1 mutant has increased test

gene expression (lacZ and 3A′ reporter systems) in the cold (Croitoru et al., 2005).

Based on this, Croitoru suggested that the R69L mutant may result in a general

increase in expression of many genes in the cell in the cold, exerting a stress on the

translational apparatus, and thus accounting for the cold sensitive phenotype. Our

polysomes data does not support this interpretation since a general increase in gene

26


expression would result in an increase in polysomes in the cold, while the opposite

is true. An alternative explanation is that the mutant IF1 results in spurious, mostly

unproductive initiation events, leading to accumulation of stalled or empty (of

mRNA or fMet-tRNAfMet) 70S ICs. At present, our data does not discriminate

whether the observed increase in the 70S peak and a decrease in polysomes in the

IF1 mutant, is due to the defective forward or reverse reaction (or both). Thus it

remains an open question that can be addressed in future studies. In any case, we

conclude from our data that the major defect of the R69L mutant IF1 is due to

defective subunit association / dissociation and not a stoichiometric imbalance of

subunits.

Figure 6. Polysome profiles of the wild type and IF1 mutant strains. Profiles from 10-40%

sucrose gradient of MG1655 (wild type) and CVR69L (IF1 mutant) strains following a

temperature downshift to 20°C. The profiles are scaled to the same relative scale; an equal amount

of material was loaded for each gradient. From unpublished results (Belotserkovsky, 2011).

2.2 Mode of action of 23S processing stem suppressors (Paper I)

In Paper I, we report the isolation of suppressor mutations that map to the processing stem

of 23S rRNA. Three independently selected mutations, two of which affected the same

position indicating their relevance, were shown to interfere with effective nucleolytic

27


processing of this structure. Indeed, we were surprised to find suppressors that were not in

either the 16S or the 23S rRNA structural genes, but reside on a transient structure that is

formed by the two terminal ends of 23S rRNA. The 23S processing stem has been well

described in literature and shown to be important for efficient maturation of the 23S rRNA

and the large 50S subunit as a whole (Allas et al., 2003; Liiv & Remme, 1998; Liiv &

Remme, 2004). In line with the fact that the processing stem is a primary substrate for the

nucleolytic enzyme RNase III – a deletion of rnc – the structural gene of RNase III, had

the same apparent suppressor phenotype as the processing stem mutants alone. It is known

that the substrate for RNase III is double stranded RNA. This enzyme has been shown to

respond to certain sequence ‘anti-determinants’, while primarily recognising structural

elements in its natural substrates (Pertzev & Nicholson, 2006; Zhang & Nicholson, 1997).

It is therefore likely that the isolated mutations in the processing stem affect the capacity

of RNase III to effectively bind (and cleave), by introducing structural distortions in the

RNA duplex, although the presence of some fully mature termini in the mutant strains

indicates that this blockage is incomplete.

While trying to understand the mode of action of these suppressors, we examined possible

scenarios were IF1 would directly interact, through its RNA chaperoning activity, with the

23S processing stem. To this extent, we compared the efficiency of 23S processing in both

the wild type and IF1 strain backgrounds, as well as at non-permissive temperatures at

which the mutant IF1 is defective. We found that there was no change in 23S processing,

forcing us to conclude that IF1 is probably not involved in any direct functional

interaction with the processing stem. Since the isolated suppressors interfere with correct

processing of 23S rRNA, we reasoned that we could detect changes in ribosome assembly

of these mutants on sucrose gradient profiles. Extensive analysis and quantification of

ribosomal subunits and 70S ribosomes based on sucrose gradient experiments indicated

that the IF1 mutant strain had increased 70S ribosomes at the expense of free subunits.

Irrespective of strain background, the suppressor mutations in the processing stem resulted

in an apparent decrease in the 50S subunits, as well as a decrease in the associated 70S

ribosomes and a concurrent increase in free subunits. Based on this data, we proposed a

model whereby the R69L mutant of IF1 is defective in ribosomal subunit association /

dissociation, while the suppressor mutations partially suppress this defect by decreasing

the pool of mature 50S subunits, thus shifting the association / dissociation equilibrium

28


towards dissociated subunits. It is worth noting that our proposed model of suppression is

compatible with the feedback regulation model of ribosome biosynthesis. In the latter, one

predicts that a stoichiometric imbalance of one subunit should be buffered out by the

appropriate adjustment of the other subunit as suggested by Nomura and co-workers

(Yamagishi & Nomura, 1988). This is because a decrease in the amount of rRNA of one

subunit would result in accumulation of ‘excess’ free r-proteins, some of which are also

regulators of synthesis of r-proteins for the other subunit. In our model, the stoichiometric

amounts of rRNA are the same for each subunit, but the assembly of 50S is retarded

relative to 30S, thus resulting in an apparent decrease in functional 50S subunits.

2.3 Mode of action of 16S rRNA suppressors (Papers II and III)

Paper II is the logical extension of previous work, whereby we describe the isolation and

characterization of further rRNA suppressors to the defective IF1. In this case,

coincidentally, all of the newly found suppressors mapped to the structural part of 16S

rRNA, suggesting a possible direct mechanisms of suppression. Suppressors were of

diverse nature and were located in the head (h32, h34 and h41), platform (h20) and

shoulder domains (h18) of the 30S subunit, with some mutations quite distant to the

binding site of IF1 (A-site). Using the same experimental approach as described in Paper I,

we determined the effect of 16S suppressors on 16S rRNA maturation, ability to support

growth, as well as behaviour of ribosomal subunits and ribosomes in the sucrose gradients.

Although the suppressors had diverse effects on growth and 16S rRNA maturation, the

common feature of all was the defect in ribosomal subunit association. In all cases, and

irrespective of strain background, the 16S rRNA suppressors gave increased free subunits

and decreased 70S ribosomes. For all but one suppressor, this was the direct result of loss

of affinity of the mutant 30S subunit for the 50S subunit, as demonstrated by in vitro

subunit association experiments. This finding was interesting in itself as none of the

suppressors were located in the known ribosomal subunit bridges. In addition, this

strengthened our initial conclusion that the major defect of the R69L mutant of IF1 was

due to defective subunit association / dissociation. It also raised the possibility that the 16S

suppressors were located in positions that are influenced by the large scale conformational

change of the 30S subunit brought about by IF1. This subject was explored in Paper III

29


(see below). In addition to effects on subunit association, a suppressor in h20 also resulted

in a slight stoichiometric imbalance of the 30S subunit relative to the 50S as judged by the

sucrose gradient data, suggesting subunit assembly defects. This paralleled the effect of

previously isolated 23S processing stem mutants hinting at a common mechanism (the

proposed model of suppression by the processing stem mutants). Interestingly, following

the publication of Paper II, a report described a functional interaction between h41 and

RNase I (Kitahara & Miyazaki, 2011). Specifically, the authors found that an intact h41 is

required to inhibit RNase I. Mutations in h41 resulted in loss of such inhibition and

consequently increased rRNA degradation. In Paper II we describe a large deletion in h41

that acts as a suppressor of mutant IF1. Although this mutant had the most extreme effect

on ribosomal subunit association, the newly found function of h41 may suggest that

another indirect mechanism, relating to RNase I activity, may also play a role in

suppression.

In Paper III we selected for mutagenesis positions in 16S rRNA that were adjacent to

those affected by the previously described spontaneous suppressors. Through non-random

mutagenesis, we derived two functional groups in 16S rRNA that interact with IF1. One of

these was located in h34 – a complex helix in the head of the subunit known to interact

with mRNA as well as contributing structurally to the A-site. Mutational analysis revealed

that complex, as yet undefined structural distortions of this helix result in the observed

suppression effect. The second functional group was also located in the vicinity of the A-

site, primarily involving the 530 pseudoknot and the adjacent helix 27. These positions

also comprise the streptomycin binding site. Some of the suppressors identified in these

positions are also known to give streptomycin resistance, and indeed we could confirm the

streptomycin resistance phenotype for two suppressors located in h27. It is known that

streptomycin resistance mutations give rise to a hyper accurate ‘open’ conformation of the

ribosome, and this has been shown previously for at least one of the newly derived

suppressors. Conversely, it is known that IF1 induces a ‘closed’ conformation of the 30S

subunit upon binding. We therefore suggested that the newly derived suppressors are

located in sites in rRNA that are effectors of the IF1 dependent conformational change of

the 30S ribosomal subunit.

30


3. Conclusions and future perspectives

This work describes a host of suppressors in 16S rRNA that could suppress the cold

sensitive IF1 mutant. Most of these suppressors occur at positions that have not been

previously implicated in IF1 interaction. These results confirm the usefulness of our

genetic approach to elucidate structure-function interactions in complex molecules.

Although the mode of action of the 23S rRNA processing stem suppressors is indirect, the

data from Paper I further highlights the importance of proper 23S processing stem

formation for maturation of 23S rRNA. It also suggests that ribosomal subunit association

/ dissociation defects can be rescued by affecting subunit maturation or assembly. This

essentially links ribosomal subunit processing and maturation to translation initiation.

The large variety and location of suppressors in 16S rRNA described in Paper II,

demonstrates that the affinity of 30S for 50S subunit can be modulated by positions other

than those in known ribosomal bridges. Paper III suggests that at least some of the

suppressors affect the overall conformational dynamics of the small subunit. Of the

outstanding questions, it would be interesting to employ in silico modelling of the effect of

h34 mutations on overall structure and dynamics of this helix. In addition, in vitro binding

studies could answer the question of whether h34 mutations affect mRNA or IF1 binding

(or both). According to our predictions, an ‘open’ conformation of the 30S induced by the

streptomycin binding site mutations (and possibly h34 mutations) should affect subunit

joining during initiation. Kinetic studies with purified mutant ribosomes and components

required for initiation could shed more light on this question. Similar studies could be

performed with the R69L mutant IF1, laying to rest the question of whether the primary

affect of the mutant is during subunit association or dissociation.

31


4. Acknowledgments

Much respect and gratitude to my supervisor Prof. Leif Isaksson, who guides with a light

but wise touch. Thank you for accepting me in your group and giving me the freedom to

explore my own interests.

Much respect and gratitude to my former supervisor Prof. Eric Dabbs, who sparked my

interest, and gave me the skills to work in the field of prokaryotic translation; and his

contribution to the idea of using second site suppressor mutations in rRNA to elucidate

structure-function interactions.

Many thanks to members of the former translation group; Victor for his positive vibes and

vino, Ernesto for wisdom and super-sharp advice, Georgina for collaboration and

discipline, Sergey and Monica for knowledge and enthusiasm.

Gratitude to Petr from Moscow State University for hosting me and schooling me on the

essence of scientific interest.

Gratitude to Eva P, Eva E for excellent administrative help and IB and Görel for technical

support and humour.

Many thanks to all at GMT for a pleasant working environment; special thanks to Morten

for the glögg parties and being a friend, Peter for helping with data in Paper II and being a

friend, Alice for silliness and being a friend, Harald for cigars and being a ‘bru’, and

Dominik, Vicky, Niklas, Hanna, Ali, Petra, Olga, Natalia, Evgenia for conversation.

32


Special thanks to Gunilla and Lisbeth for continued support and friendship in Sweden. To

my friend in Germany – Fabian, sorry about the timing and congratulations!

Much love and gratitude to Jim and May – hope to visit soon one of these days!

Shouts to my long time mates for the many shared memories back from the good’ol -

Connaire and Chris.

Much love to my ‘GF’ - Tove for putting up with me (there goes the 2-year rule!); my

gratitude to her family for accepting me.

Much love to my family; my sister for support and my parents for doing everything for

me.

33


5. References

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