Functional Studies of Escherichia Coli Stringent Response Factor Rela

Home , Alarmone, Diphosphotransferase, Guanosine pentaphosphate, Stringent response

Functional Studies of Escherichia coli Stringent Response Factor RelA

Ievgen Dzhygyr

Department of Molecular Biology, MIMS Umeå 2018 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD Copyright © Ievgen Dzhygyr ISBN: 978-91-7601-934-4 ISSN: 0346-6612; New series No: 1970 Front cover design: Ievgen Dzhygyr Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU Print Service, Umeå University Umeå, Sweden 2018

To my parents/Моїм батькам

Table of Contents

Abstract ...... ii Abbreviations ...... iv Papers included in this thesis ...... vi Introduction ...... 1 Background ...... 1 Synthesis of (p)ppGpp ...... 2 Effects of (p)ppGpp on DNA replication ...... 4 (p)ppGpp mediated regulation of transcription ...... 5 Effects of (p)ppGpp on GTPases ...... 7 Effects of (p)ppGpp on Bacillus subtilis metabolism ...... 9 RSH domains organization and classification ...... 11 Structure of HD and SYNTH domains of long RSHs ...... 13 Role of the stringent response in bacterial virulence ...... 19 Aims of the study ...... 24 Results and Discussion ...... 25 Role of RelA and ribosomal structural elements in RelA activity (Paper I and II)...... 25 Allosteric regulation of RelA by (p)ppGpp (Paper I and II) ...... 27 RelA interaction with tRNA off the ribosome (Papers I and II) ...... 29 Effect of translational inhibitors on RelA induced stringent response (Paper III) .... 30 Conclusions ...... 34 Acknowledgements ...... 35 References ...... 36

Abstract

RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation of Escherichia coli to nutritional stress such as amino acid deficiency. It detects the deficiency of amino acids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is charged with amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to produce alarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is a global metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolic processes, such as DNA replication, gene expression, cell wall synthesis, and translation. These changes also include activation of different virulence factors and are proposed to drive formation of a bacterial sub-population that is highly resilient to antibiotic treatment, the so- called persisters.

For a long time the molecular mechanism of RelA’s interaction with the ribosome-deacylated tRNA complex, which leads to its activation, was unknown. Only recently several cryo-EM structures of RelA-ribosome complex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNA complex. Guided by these structures we investigated the role of RelA’s domains in this interaction by constructing a set of RelA C-terminal truncates and subjecting them to biochemical and microbiological experimentation. These experiments were complemented with mutations in ribosomal RNA at positions that interact with RelA, namely A-site finger, thiostrepton loop, and sarcin-ricin loop.

We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex, whereas, the set of truncated proteins missing either one, two or three C-terminal domains do not respond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncated versions can still be activated by vacant 70S ribosome as well as pppGpp, though to lesser extent than wild type RelA. This suggests that an allosteric regulation site for (p)ppGpp is located at the N-terminal domain of RelA and that truncate which lacks C-terminal domain can still interact with the ribosome. The mechanism of this interaction is yet to be elucidated.

We have shown that A-site finger of the ribosome is required for RelA activation and recruitment to the ribosome. Using EMSA and biochemical assays, we have shown that RelA and deacylated tRNA do not form a stable complex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylated tRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.

Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also tested several strategies for RelA inhibition by antibiotics, which target ribosomes and the interaction between RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostrepton inhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. ppGpp production is also inhibited by chloramphenicol and tetracycline.

iii

Abbreviations

ACP Acyl carrier protein ACT Aspartate kinase-chorismate mutase-TyrA ASF A-site finger ATP Adenosine triphosphate C3HB Central 3 helix bundle CTD C-terminal domain DAP Diaminopimelic acid DMSO Dimethylsulfoxide DNA Desoxyribonucleic acid EF-G Elongation factor G EF-Tu Elongation factor, thermounstable GDP Guanosine diphosphate GMK Guanosine monophosphate kinase Guanosine-5'-triphosphate,3'-diphosphate GppA pyrophosphatase GTP Guanosine triphosphate HD Hydrolase domain HPRT Hypoxanthine phosphoribosyltransferase IF2 Initiation factor 2 IMP Inosine monophosphate IMPDH Inosine monophosphate dehydrogenase LPS Lipopolysaccharides NDK Nucleoside diphosphate kinase NTD N-terminal domain NTP Nucleotide triphosphate PDE Phosphodiesterase PPi Inorganic diphosphate ppGpp Guanosine tetraphosphate pppGpp Guanosine pentaphosphate (p)ppGpp Guanosine tetra- and pentaphosphate collectivelly PRPPP Phosphoribosylpyrophosphate RF Recycling factor RNA Ribonucleic acid RNAP RNA polymerase

ROS Reactive oxygen species RRM RNA recognition motif SAH Small alarmone hydrolase SAS Small alarmone synthase SC "Starved" complex SYNTH Synthase domain TGS Threonyl-tRNA-synthase, GTPase, SpoT tRNA Transfer RNA XMP Xanthosine monophosphate XMPGAT XMP-glutamine amidotransferase ZFD Zn-finger domain

Papers included in this thesis

I. Dzhygyr I and Hauryliuk V (2018). Cut-back analysis reveals the role of RelA's individual domains in its activation by 'starved' ribosomal complexes and pppGpp alarmone nucleotide. (manuscript)

II. Dzhygyr I*, Kudrin P*, Ishiguro K, Beljantseva J, Maksimova E, Oliveira SRA, Varik V, Payoe R, Konevega AL, Tenson T, Suzuki T, Hauryliuk V. (2018). The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA. Nucleic Acids Research, 46(4):1973-1983.

III. Kudrin P, Varik V, Oliveira SR, Beljantseva J, Del Peso Santos T, Dzhygyr I, Rejman D, Cava F, Tenson T, Hauryliuk V. (2017). Subinhibitory oncentrations of Bacteriostatic Antibiotics Induce relA- Dependent and relA-Independent Tolerance to β-Lactams. Antimicrobial Agents and Chemotherapy, 61(4), 1–17.

* denotes equal contribution

Introduction

Background

The way bacteria regulate their metabolism in response to signals from inside the cell and the environment is fascinating in its complexity and efficiency. In order to sustain life, thousands of chemical reactions are happening in the cell simultaneously in highly coordinated fashion. Bacterial cells need to sense a multitude of different environmental cues such as presence of nutrients, minerals, and factors secreted by other bacteria. Rapid adjustment of the cell metabolism according to such signals is crucial for the survival of the bacteria, since evolutionary pressure forces living organisms to manage their resources in the most efficient way. In order to achieve such efficiency bacteria have developed a number of molecular mechanisms which allow for sensing changes in surrounding, rapid transmission of these signals into the cell and execution of specific metabolic pathways.

One of the mechanisms that bacteria employ to adjust their gene expression program to external stimuli such as availability of nutrients is the stringent response (SR). Initially the stringent response was discovered as a rapid drop in production of stable types of RNA in response to amino acid deficiency (1,2). It is executed via effector (alarmone) nucleotides guanosine tetra- and pentaphosphate (ppGpp and pppGpp, respectively) and regulates several key cellular processes such as DNA replication (3,4), transcription initiation (4,5), degradation and synthesis of lipids and phospholipids (6–8), transport of nucleobases across cell membrane (9) and nucleotides synthesis (10), amino and fatty acids synthesis and transport across membrane, proteolysis (11–13), and cell wall synthesis (12,14) as summarized in Figure 1. The stringent response also regulates host-pathogen interaction for many clinically significant microorganisms. It is an essential element of bacterial virulence (15), fitness (8), and antibiotic tolerance (16–18).

Figure 1. (p)ppGpp-mediated stringent response in bacteria

Nutritional stress is sensed via RSH proteins that produce (p)ppGpp, the mediator of the stringent response in bacteria. It leads to downregulation of main cell processes directed to sustain rapid cell growth and replication and activates stress coping mechanism.

Synthesis of (p)ppGpp

In Escherichia coli level of stringent response alarmone molecule (p)ppGpp is maintained by two enzymes – RelA and SpoT – which gave the name to the family of homologous proteins from other organisms, RSH (RelA SpoT Homologues; [19,20]).

RelA is a monofunctional multi-domain ribosome-associated protein. It interacts with the ribosome and tRNA at the acceptor site of the ribosome (A-site) and monitors the aminoacylation status of the A-site tRNA (Figure 2A). When the cell experiences amino acid starvation and intracellular amino acid level drops, the amount of deacylated tRNA increases. When RelA detects such deacylated tRNA it starts to synthesize (p)ppGpp using ATP and GDP or GTP as substrates. The enzyme transfers two phosphate groups (β and γ) from ATP molecule to 3´hydroxyl of ribosose of GDP or GTP molecule resulting in production of tetra- and pentaguanosinephosphate respectively (21).The general scheme of (p)ppGpp synthesis and enzymes involved in it is represented in Figure 2B.

Despite the fact that RelA has a slight preference for GDP (22), the main product of RelA during the stringent response in E. coli is pppGpp. This happens because the concentration of GTP in the cell is around 5 times higher than GDP (23). After synthesis, the pppGpp molecule is further processed by guanosine pyrophosphatase GppA (24), which removes one phosphate group from 5’ end of ribose converting pppGpp into ppGpp. It was shown that both pppGpp and ppGpp can allosterically upregulate RelA activity, with pppGpp being stronger activator than ppGpp (22,25).

Figure 2. Schematic of RelA activation and (p)ppGpp metabolism in E. coli (modified from [23])

A) During amino acid abundance elongation factor EF-Tu brings acylated tRNA to the ribosome. During amino acid deficiency, the pool of acylated tRNA decreases and translation stalls. At this conditions deacylated tRAN and RelA can bind the ribosome and (p)ppGpp synthesis starts. B) In E. coli (p)ppGpp is synthesized by two enzymes: monofunctional enzyme RelA, which has strong synthase and no hydrolase activity, and bifunctional enzyme SpoT with weak synthase and strong hydrolase activity. Enzyme guanosine pyrophosphotase (GppA) converts pppGpp to ppGpp.

During the stringent response ppGpp concentration increases from the base level of 30-50 µM to 800 µM (23). In order to be able to go back to rapid growth mode when the environmental conditions become favorable, bacteria should be able to hydrolyze (p)ppGpp back to its base level. In case of E. coli, this function lies on another RSH, SpoT, which hydrolyses (p)ppGpp to GTP or GDP and PPi (19,27,28). Hydrolase activity of SpoT is essential for E. coli survival because mutants with deletion of spoT gene are not viable if relA gene is functional (29).

Besides hydrolyzing (p)ppGpp, SpoT has weak synthase activity and can synthesize (p)ppGpp in response to stresses other than shortage of amino acids (19,30). The factors which trigger SpoT synthase activity are carbon source deficiency (31,32), iron deficiency (33), fatty acid starvation or phosphate starvation (34). However, the mechanism of SpoT activation is not clear yet. It was shown that in case of fatty acid starvation SpoT could be activated by interaction with a key protein in fatty acid biosynthesis, acyl carrier protein (ACP) and that the proteins interact through TGS (Threonyl-tRNA-synthase, GTPase, SpoT) domain of SpoT, see Figure 3, (35,36).

Figure 3. Proposed mechanism of SpoT activation by acyl carrier protein (modified from [33])

During fatty acid starvation acyl carrier protein (ACP) binds to TGS domain of SpoT which leads to inhibition of hydrolase (HD) domain and activation of synthase (SYNTH) domain, thereby increasing production of (p)ppGpp.

Effects of (p)ppGpp on DNA replication

(p)ppGpp down regulates DNA replication by inhibiting primase DnaG (5,37) which is responsible for synthesis of short RNA primers required for transcription initiation. In this case (p)ppGpp acts as an analogue of GTP. It binds to the active site of the primase and stalls the reaction, since it cannot be incorporated in the primer as GTP (4,5).

(p)ppGpp mediated regulation of transcription

As a master regulator, (p)ppGpp affects main biochemical processes in the cell which allow for rapid adjustment of whole metabolism with respect to environmental conditions. During amino acid starvation gene expression changes in such a way that genes involved in anabolism processes such as ribosome synthesis, DNA synthesis and cell wall synthesis are inhibited, at the same time transcription from stress genes and genes responsible for amino acid synthesis and uptake are upregulated (12,18).

Several mechanisms were proposed how (p)ppGpp may affect transcription. According to structural studies (3,38,39) and cross-linking experiments (40) (p)ppGpp can bind RNA polymerase (RNAP), the enzyme responsible for mRNA synthesis on DNA matrix, at several sites. One of these sites is located at the interface of RNAP subunits β´, α and ω at a distance of 28 Å from the active site (3), Figure 4. Upon binding (p)ppGpp restricts ratcheting movement of RNAP domains, and affects the catalytic activity of the enzyme. This infers that during the initiation step, unstable RNAP-DNA open complexes, for which fast initiation is required, will collapse, while more stable complexes can still initiate transcription (3).

Transcriptional factor DksA is another key player in transcription regulation during the stringent response. It binds near the secondary channel of RNAP and contributes to the formation of the second ppGpp binding site (39,40). Binding of DksA to RNAP may influence the ratcheting movement of RNAP domains (41) or RNAP promoter sequence interaction (40) which may augment the ppGpp effects, both positive and negative, on transcription from different promoters (42,43). In some cases the effects of DksA and (p)ppGpp can be opposite (44,45).

β’ β

DksA ω

ppGpp binding site 2 ppGpp binding site 1

Figure 4. Binding sites of ppGpp at RNA polymerase

First binding site for ppGpp (blue) is located at the interface between β’ (transparent gray) and α (rosy brown) subunits. The second site is located at the interface between RNAP and DksA (red). The tip of DksA is reaching the active site of RNAP. Binding of ppGpp at the first site imposes steric clashes for proper movement of RNAP subunits during RNA synthesis. ppGpp binding to the second site can facilitate DksA binding to RNAP and enhance its effect. The other RNAP subunits colored: β (tan), ω (orange), σ (brown). Active site of RNAP contains Mg2+ ion (black sphere). Crystal structure presented as per (39); PDB accession number 5vsw.

According to another model (Figure 5) of transcription regulation during the stringent response, ppGpp may regulate transcription from different promoters through altering affinity of the core RNAP enzyme to housekeeping transcription initiation factor σ70, responsible for promotor recognition (46,47). This allows other σ factors to bind to RNAP and guide transcription of stress response genes rather than housekeeping genes, which are transcribed during growth in optimal conditions (44,46–48).

Figure 5. Schematics of interaction between RNAP and sigma factors in presence of ppGpp (modified from [42])

During favorable conditions σ70 factor guides RNAP to transcribe genes from σ70 promoters. During the stringent response, binding of ppGpp to RNAP leads to conformational changes which destabilize RNAP interaction with σ70 factor and allow for recruitment of other σ factors responsible for transcription of stress response genes from different promotors.

Effects of (p)ppGpp on GTPases

GTPases are a class of enzymes that are powered by tightly controlled GTP hydrolysis. (p)ppGpp, acting as an orthosteric competitor of the GTP substrate, acts as an inhibitor of different GTPases involved in numerous cellular processes, notably translation and ribosome biogenesis (50).

Translational GTPases targeted by (p)ppGpp are initiation factor 2 (IF2), elongation factor Tu (EF-Tu), and elongation factor G (EF-G) (51). In case of initiation factor 2 (IF2), which brings initiatory tRNA charged with formylmethionine to 30S ribosomal subunit and positions it at the peptidyl site (P-site) of the ribosome, the phosphate groups of (p)ppGpp at 3´position of ribose can cause steric hindrance and inhibit interaction between IF2 and ribosome or initiatory tRNA (52,53). (p)ppGpp was also suggested to inhibit translation through release factor 3, RF3 – a factor that facilitates dissociation of release factors 1 and 2 from the ribosome (54). It was shown that ppGpp inhibits this process (55).

GTPases involved in ribosome assembly targeted by (p)ppGpp include BipA (TypA), Obg (CgtA, YhbZ), RsgA, RbgA, Era, and HflX (56,57). During stringent response BipA regulates expression of virulence factors and biofilm formation in different eubacteria (58,59). When the level of (p)ppGpp increases, BipA alternates its affinity from large to small ribosomal subunit (60). This may happen due to the fact that binding of (p)ppGpp causes steric hindrance between BipA and large ribosomal subunit (61).

The exact role of RsgA, Era, HflX and RbgA are unknown (56). However, mutant lacking these proteins show defect in ribosome assembly (62,63). Rsg and Era were shown to bind to 30S subunit while HflX and RbgA bind to 50S subunit (56). Non-hydrolysable analogues of GTP cause the proteins to stay bound to ribosomal subunits (62,64,65). It was speculated (56) that in case of inhibition of GTPase activity by ppGpp, GTPases remain in bound state and may prevent subunits association (66). Therefore, increase in (p)ppGpp level leads to decrease in number of ribosome and causes inhibition of translation.

Obg (CgtA, YhbZ) is a ribosome associated GTPase conserved among bacteria and chloroplasts (67). The exact function of Obg is not clear. However, it is essential for bacterial growth and stress response (68,69). The protein binds (p)ppGpp and takes part in mediation of the stringent response in E. coli (70) and B. subtilis (71). It also was shown that CgtA may interact with SpoT during normal growth conditions (72,73).

(p)ppGpp may also regulate nucleotide salvage pathway by inhibiting YgdH (57), a GTPase which degrades monophosphate nucleotides to ribose-phosphate and a nucleobase.

Effects of (p)ppGpp on Bacillus subtilis metabolism

In Bacillus subtilis, a model organism from Firmicutes phylum, regulation of the gene transcription during the stringent response is not mediated through RNAP but rather through depletion of GTP pool (74). This happens due consumption of GTP during the reaction of (p)ppGpp synthesis and decrease in GTP synthesis through inhibition of enzymes involved in it. (p)ppGpp inhibits guanosine monophosphate kinase (GMK), hypoxanthine phosphoribosyltransferase (HPRT), and inosine monophosphate dehydrogenase (IMPDH) (75).

In bacteria, GTP is synthesized via several consecutive reactions either through de novo pathway or through salvage pathway. As described in (76), in case of de novo synthesis phosphoribosylpyrophosphate (PRPP) is converted to inosine monophosphate (IMP) through a number of steps in which purine ring is synthesized on phosphoribose base. IMP is converted to xanthosine monophosphate (XMP) by inosine monophosphate dehydrogenase (IMPDH). XMP is converted to guanosine monophosphate by XMP-glutamine amidotransferase. Then guanosine monophosphate kinase (GMK) adds phosphate residues converting GMP into GDP. In its turn GDP is converted to GTP by nucleoside diphosphate kinase (NDK), see Figure 6.

Figure 6. Inhibition of GTP synthesis in B. subtilis by (p)ppGpp (modified from [71])

Stringent response in B. subtilis is induced through depletion of GTP pool during alarmone synthesis and through the inhibition of key enzymes involved in GTP de novo synthesis and salvage pathways by (p)ppGpp. (p)ppGpp inhibits guanosine monophosphate kinase (GMK) and hypoxanthine phosphoribosyltransferase (HPRT). Depletion of GTP pool leads to inhibition of transcription of inosine monophosphate dehydrogenase (IMPDH) through inhibition of transcription regulator CodY, which activates it when GTP level is high.

In case of salvage pathway, GTP is synthesized from the products of GTP metabolism hypoxanthine and guanine. Hypoxanthine is converted to IMP by hypoxanthine-guanine phosphoribosyltransferase (HPRT), and guanine is converted into GMP by the same enzyme. The key enzymes affected by (p)ppGpp vary between species (20). In E. coli, (p)ppGpp inhibits IMP dehydrogenase (77,78) while in B. subtilis the main targets are GMK and HPRT (75).

Another mechanism of gene transcription regulation, which is triggered in firmicutes when GTP level decreases, involves CodY (10), which is a master regulator protein in Gram-positive bacteria (Figure 7). This transcriptional repressor which regulates expression of more than 100 genes in B. subtilis is inhibited by high concentration of GTP and gets activated only when the level of GTP drops down (79).

Figure 7. Gene regulation in B. subtilis (modified from [10])

Transcription of genes required for amino acid biosynthesis( BCAA, methionine, leucine, threonine) is repressed by both GTP level and CodY regulatory protein. During stringent response when GTP level goes down these genes are activated.

RSH domains organization and classification

RSH proteins comprise a big family of proteins which are present in bacteria, plants, and animals (30). Atkinson et al. divided bacterial RSHs in 3 major groups. Firs is comprised of long RSHs including bifunctional Rel, RshA-D, SpoT and monofunctional RelA. Rel proteins are commonly found in α, δ, and ε proteobacteria, firmicutes, and cyanobacteria (Figure 8). SpoT are common in β and γ proteobacteria, while RshA-D in δ proteobacteria, bacilli, actinobacteria, archaeplastida, bacteroides, and haptophycaea. Monofunctional RelA proteins, which lack hydrolase activity, are common in β and γ proteobacteria. Next group is represented by small monofunctional alarmone synthases (SAS), common in proteobacteria, firmicutes, archaea, and actinobacteria. The other group is small alarmone hydrolases (SAH), capable only of (p)ppGpp hydrolysis. SHSs are also present in proteobacteria, firmicutes, archaea, and in some animals.

Long RSHs have common domain organization (Figure 8). In general they comprise of hydrolase, synthase, TGS (Threonyl-tRNA-synthase, GTPase, SpoT domain), Zinc Finger Domain (ZFD) known also as Conserved Cysteines domain (CC), and ACT domain (Aspartate kinase-Chorismate mutase-TyrA, prephenate dehydrogenase) (30), which was also classified as RNA recognition motif domain (RRM) (80). Some RSHs can have calcium binding domain (EF-hand), other can miss one or two C-terminal domains (30,50).

RSH with variations in domain organization are also found in plant chloroplasts (30). Among α, δ and ε proteobacteria the most common type of RSH is bifunctional long RSHs that are present in combination with short RSH, SAS enzymes. However, in β and γ proteobacteria, one of the RSHs is a bifunctional enzyme with a weak synthase and strong hydrolase function and the other is a monofunctional long RSH that can perform only synthesis of (p)ppGpp, e.g. E. coli (30,50). SAS and SAH distribution between bacteria varies greatly. General rule of RSHs distribution among bacteria is that synthase activity should be balanced by hydrolase activity in form of either bifunctional enzymes or a combination of bifunctional and several monofunctional enzymes. However, in case of animals and fungi, small RSHs responsible either for synthesis or hydrolysis of (p)ppGpp can be present alone (30). In these cases the production of (p)ppGpp is compensated by other enzymes which are able to hydrolyze the alarmone (30). The role of standalone SAHs is not completely understood. Obligate intracellular bacteria from Chlamydiae, Planctomycetes Verrucomicrobia phyla do not possess any RSHs (30).

It was suggested (30) that having an arsenal of RSH proteins may increase the stability of bacterial stringent response system and make it more robust and insensitive to stochastic fluctuations in signal levels and protein expression levels. Ability to express long and short RSHs in response to the cell’s needs may give a capacity to adjust metabolism to environmental challenges in a more precise way.

Figure 8. General domain structure of bacterial RSHs and their distribution among classes (modified from [28])

A) Long RSHs are comprised of N-terminal hydrolase (HD) and synthase (SYNTH) domains both active in case of Rel, SpoT, and RshA-D and with inactive HD in case of RelA. The absence of hydrolytic activity of RelA is compensated by presence of SpoT. C-terminal part of long RSHs commonly contains TGS, CC (ZFD), ACT (RRM) domains. B) Long monofunctional enzymes (RelA) in pair with bifunctional (SpoT) are common among β, γ proteobacteria (brown line). α, δ and ε proteobacteriav (blue line) contain bifunctional RSHs. SAS and SAH enzymes may be present in different groups of bacteria in combinations with other RSHs.

Structure of HD and SYNTH domains of long RSHs

Due to the tendency of RSH proteins to aggregate at low concentrations (7-8 µM in the case of E. coli RelA ) so far there were no successful cases of full-length RelA crystallization and, until recently, full structure of the protein was unavailable. Some clues on how RSHs work were aquired by solving the structure of N-terminal part of bifunctional Rel from Streptococcus equisimilis (RelSeq), which was done by Hogg and colleagues (81). They obtained, the structure of truncated, 385 amino acid residues long RSH which contains only the hydrolase and synthase domains (Figures 10-12).

Figure 10. Crystal structure of N-terminal part of RelSeq from Streptococcus equisimilis

N-terminal part (1-385) of bifunctional RelSeq consists of synthase domain (blue), hydrolase domain (purple) and central 3 helix boundle (C3HB) between them (pink). C3HB acts as a hinge and enables

RelSeq to switch between hydrolase ON/synthase OFF or hydrolase OFF/synthase ON states. Crystal structure presented as per (81); PDB accession number 1vj7.

The structure of RelSeq revealed that hydrolase domain consists of 3 α-helices which form a bundle. This entral 3 helix bundle (C3HB) connects hydrolase and synthase domains. HD domain and C3HB form a fold, which is common for phosphodiesterase (PDE) catalytic domain. PDE is an enzyme involved in degradation of other messenger molecules such as cGMP and cAMP. The synthase domain of RelSeq is composed of 4 α helices and 5 antiparallel β sheets (81).

Hydrolase and synthase domains change their conformation in such a way that only one of the domain can be active at a time. Binding of (p)ppGpp to the hydrolase site locks it in the active conformation, thus disabling (p)ppGpp synthesis (81,82).

Figure 11. Structure of synthase domain of RelSeq from S. equisimilis

The crystal structure shows the synthase domain of RelSeq with GDP (black) as the substrate in the middle. Amino acid residues involved in GDP and ATP coordination are shown in yellow. The structure is presented as per (81); PDB accession number 1vj7.

According to (81), HD’s active site (Figure 12) is composed of Lys45, Leu155, Arg44, Asn148, Thr151 as well as Ser46. These residues are conserved in bifunctional RSH enzymes but are mutated in monofunctional RSHs (81). HD’s active site contains also Mn2+ ion coordinated by His53, His77, Asp78, Asp144, which are conserved in bifunctional RSHs. Both Glu81 and Asp82 may be involved in activation of water molecule for the catalytic reaction of dephosphorylation (81)

Figure 12. Structure of hydrolase domain of RelSeq from S. equisimilis

The crystal structure shows the hydrolase domain of RelSeq with cyclic ppG2’:3’p (black) as the substrate in the middle. Amino acid residues involved in its coordination are shown in yellow. Mn2+ essential for hydrolysis is represented by purple sphere. The structure is presented as per (81); PDB accession number 1vj7.

Charge reversal in RelA’s SYNTH domain

Bifunctional and monofunctional RSHs show difference not only in HD but also in SYNTH domain organization. It was shown by Sajish and colleagues (83,84) that SYNTH domain of bifunctional RSHs have three conserved residues RXKD which are likely to be involved in Mg2+ binding and substrate coordination in the active site. The presence of this conserved sequence causes inhibition of synthase activity of bifunctional RSHs in conditions where Mg2+ is in excess (83,84). This inhibition is absent in monofunctional RSHs such as E. coli RelA where positively charged Arg and Lys are substituted with negatively charged Glu and Asp respectively forming EXDD sequence. This sequence is conserved among long monofunctional RSHs (84).

In the article (84) authors speculate that in bifunctional RSHs binding of Mg2+ causes conformational changes (loop-to-helix transition) in the domain. The helix formed blocks the active site thereby preventing substrate binding. In case of moofunctional RSHs, EXDD forms an extra binding site for the magnesium ion and prevents conformational changes, therefore, synthase activity is not inhibited when Mg2+ is in excess.

Activation of RelA and Rel by ‘starved’ ribosomal complexes

Though it was known from the 60s that long RSHs could interact with both ribosome and A-site tRNA, and that C-terminal domain of RSHs is involved in this interaction, the exact position of the protein on the ribosome was unclear (85–87). Only recently cryo-electron microscopy (cryo-EM) structures of full length E. coli RelA in complex with the ribosome and uncharged tRNA have been solved, detailing the exact RelA location on the ribosome and has revealed details of its interaction with ribosomal structural elements (80,88,89), see Figure 13. Cryo-EM structures show that, RelA is wraped around deacylated tRNA in the acceptor site. The deacylated tRNA upon contacting RelA takes unusual A/R conformation where its acceptor part is bent out from peptidyl transfer center.

Deacylated tRNA in the A-site is recognized by RelA’s TGS domain. This interaction involves residues Arg487, Lys491, Lys498, Arg489, His493, Arg497. Residues Arg438, His432 interact with CCA end of tRNA. ZFD of RelA interacts with A-site finger of the ribosome, namely helix 38 (80,89). RelA ineracts with S13 and S19 proteins of small subunit depending on the ratcheting state of the ribosome (80). Interestingly enough, no interaction was detected between L11 protein of large subunit and RelA though it was shown before that L11 protein is crucial for RelA activation by deacylated tRNA in the A-site (86). From the structures it looks that L11 takes part in tRNA-RelA complex accommodation on the ribosome but only the tRNA interacts with it (80,89).

Loveland and colleagues have also shown that synthase domain of RelA is located in close proximity to spur of small subunit which infers possible role of the spur in RelA activation. As a supporting argument, authors regard a structure of metazoan innate immune sensor OAS1, which resembles synthase domain’s structure and is activated upon binding to double stranded RNA (89).

It is proposed that RelA can adopt two conformations, a closed one when the synthesis of (p)ppGpp is inhibited and an open conformation when (p)ppGpp synthesis is active (90,91). Upon binding the ribosome-deacylated tRNA complex RelA adopts the open conformation and becomes fully active (91). For bifunctional RSH from Mycobacterim tuberculosis the presence of deacylated tRNA itself may promote increase in (p)ppGpp synthesis by decreasing KM of the reaction for GTP (92,93). This may imply that deacylated tRNA can stabilize RSH in the open conformation promoting increase in activity. However, whether the effect is common for all long RSHs or only for bifunctional enzymes is still an open question.

50S

P E

A RelA

30S

Figure 13. Structure of RelA bound to the ribosome

RelA is wrapped around A-site tRNA (goldenrod) with the RRM (dark red) and the ZFD (blue) domains interacting with ribosomal A-site finger while the TGS domain (dark green) interacts with the tRNA. Synthase (orangered) and hydrolase (dark blue) domains of RelA are located outside the ribosome. CTD and NTD domains are connected by a linker (magenta). P-site and E-site tRNAs are olive green and rosy brown respectively, mRNA is light blue. 50S ribosomal subunit is colored yellow, 30S subunit is colored pale blue. Cryo-EM structure is presented as per (80); PDB accession number 5IQR.

Role of the stringent response in bacterial virulence

In many bacteria expression of virulence factors is connected to general stress response (15). Among most important virulence factors regulated by (p)ppGpp in different bacteria are adherence, quorum sensing, biofilm formation, antibiotic tolerance, ability to invade host tissues and host cells, ability to survive inside macrophages, secretion systems, motility and sporulation (15).

In enterohemorrhagic E. coli (EHEC), expression of the locus of enterocyte effacement (LEE) genes which encodes for adherence factors and factors required for host tissue colonization, is activated by ppGpp in response to harsh conditions in the lower intestine (94). In uropathogenic E. coli (p)ppGpp activates transcription of fim promotor genes which encodes for type I fimbriae (adhesion factors)(95).

In Pseudomonos aeruginosa, the causative agent of cystic fibrosis, (p)ppGpp regulates quorum sensing genes (QS), biofilm formation genes, proteases and toxin production, and swarming, necessary for virulence (96–99).

Yersinia pestis carries different virulence factors on 3 plasmids, which are required to survive in different hosts and cause infection. ppGpp is essential for regulation of the gene expression from these plasmids (15). ppGpp stimulates production of type three secretion system T3SS such as YopE and YopH. It also promotes expression of LcrV factor responsible for inhibition of proinflamatory host factors e.g. tumor necrosis factor and interferon gamma and stimulation of interleukin 10 release (100).

It was shown that (p)ppGpp increases Vibrio cholera antibiotic tolerance via reducing negative effect of antibiotic induced ROS (Reactive Oxygen Species) production, presumably through regulation of iron metabolism (101). Expression of haemagglutinin protease (HAP), an important virulence factor at later stages of infection, requires ppGpp and DksA (102). (p)ppGpp also stimulates production of ToxR and ToxT, proteins which orchestrate expression of virulence genes in V. cholera (103).

Salmonella enterica, in order to infect the host and to be able to survive inside different host tissues, employs several sets of virulence genes from Salmonella Pathogenicity Islands (SPI1 and SPI2). The expression of these genes requires ppGpp production and dksA gene expression (104,105). It was shown that ppGpp mediates virulence factor expression by interacting with S. thyphimurium transcription regulator protein SlyA, facilitating its transcription,dimerization, and promotor binding (106). SlyA is one of the regulatory proteins which initiates activation of SPI2 operon in response to stressful environmental factors such as low pH and Mg2+ , conditions which Salmonella encounters inside the phagosome of macrophages (106).

Francisella tularensis can infect a broad variety of hosts and cause tularemia in humans. The disease has high infectivity and incapacitating ability (107). In F.tularensis ppGpp regulates expression of Francisella pathogenicity island (FPI) genes, essential for survival inside machrophages (108). ppGpp promotes interaction between PigR regulatory protein and MglA-SspA complex which guides RNAP to the promoter of FPI genes (109).

During infection, Mycobacterium tuberculosis can switch to a latent state characterized by low growth rate and increased tolerance to antimicrobial agents. It was shown that bifunctional Rel plays a key role in bacterial survival in mice (8). (p)ppGpp is also required for expression of virulence genes (110). It also affects antigen variation in bacterial cell wall, altering host immune response (111).

Other species, which raise significant concern for the public health, also employ (p)ppGpp for regulation and adapting their metabolism to a variety of conditions in the environment and inside the host. Among them are pathogens such as Enterococcus faecalis (112,113), Shigella flexneri (114), Legionella (115,116), Listeria monocytogenes (117), Staphylococcus aureus (118,119), Streptococci (120,121), and Helicobacter pylori (122,123).

Role of (p)ppGpp in antibiotic tolerance and formation of persister cells

In addition to virulence factor transcription regulation, high (p)ppGpp level was reported to increase antibiotic tolerance in several bacteria, among which are E. coli (16,124) S. aureus (125,126), E. faecalis (127), B.subtilis (128), and P. aeruginosa (129,130).

Formation of a pool of bacteria, which don’t have resistance factors in their genome but can tolerate antibiotic treatment, so called persisters, is not a function of a single gene or molecule. There can be several mechanisms which may lead to this phenomenon (reviewed in [124]). In general, bacterial population contains a small percentage of persistent cells (Figure 14). Stress response, which slow down cell metabolism, facilitates appearance of resistant bacterial subpopulation (16,132). This results in ineffectiveness of antimicrobial agents to cause damage to vital cell processes, since these processes are put on halt or their rate is significantly reduced (131). Taking into account the role of (p)ppGpp in mediating stress response it can be viewed as one of the important players in this process (133).

(p)ppGpp can contribute to antibiotic tolerance in several ways. One way is the inhibition of activity of biological processes, which are targets for the antibiotics. Elevated levels of (p)ppGpp endow tolerance to β-lactams (17,124,134), antibiotics which affect natural balance between bacterial cell wall synthesis and degradation eventually causing its destruction. ppGpp inhibits synthesis of cell wall components such as peptidoglycan (17). The same applies to antibiotics which inhibit translation or DNA replication. (p)ppGpp slows down these cell functions (16,130,135). (p)ppGpp can also contribute to increased antibiotic tolerance through induction of the expression of efflux pumps which pumps out antibiotics from the cell. This protective mechanism was documented in E. coli, Salmonella and Schigella for microcin J25 (136).

Figure 14. Response of different bacterial population to antibiotic treatment (modified from [129])

During antibiotic treatment, resistant mutants continue to grow in presence of antibiotic (blue solid line). In case of wild type (WT, black solid line), the CFU/ml drops down drastically, since WT population mainly consists of antibiotic sensitive bacteria (black double dotted line). However, a small part of the WT population is represented by a persistent population (black dashed line), which can survive antibiotic treatment.

Antibiotics targeting the stringent response

The profound effect of (p)ppGpp level on major cell processes and its role in increasing antibiotic tolerance and persister formation suggests that drugs which target stringent response would be of great interest for treatment of bacterial infections in order to prevent formation of persistant population of bacteria.

At present several compounds are proposed as specific inhibitors of RelA. One of them is Relacin. Structure of Relacin mimics the structure of (p)ppGpp (Figure 15). This allows Relacin to bind and block the active site of RelA (138). Relacin was shown to inhibit spore formation in B. subtilis and B. anthracis, and also affect biofilm formation by B. subtilis (138).

Figure 15. Structures of Relacine and pppGpp (138)

Relacin mimics (p)ppGpp structure. In Relacin phosphate groups at positions 3’ and 5’ are replaced by glycyl-glycine dipeptides attached to ribose through carbamate link .

Another antimicrobial agent claimed to act against (p)ppGpp accumulation is a short peptide 1018 (VRLIVAVRIWRR-NH2). It was shown to prevent biofilm formation in a number of pathogens when used at low concentrations (0.8 μg/mL) and kill bacteria at higher concentrations (10 μg/mL). According to authors it binds (p)ppGpp which leads to degradation of the alarmone (139).

However, the effect of Relacin on B. subtilis and 1018 on E. coli, B. subtilis and P. aeruginosa was found to be controversial (140,141). In the study by Andresen et al. (141) the reverse sequence (RRWIRVAVILRV-NH2) of polypeptide showed same anti-biofilm properties as the original 1018. This suggests that cationic nature of the peptide rather its interaction with RSHs determines its effectivity. Both of the antimicrobial agents showed no specificity towards RelA or (p)ppGpp being equally efficient against WT and ppGpp0 strains (140,141).

Aims of the study

The focus of this thesis is on the role of RelA and ribosomal structural elements in RelA activation and regulation of its activity during the stringent response. We aimed to:

 establish the role of ribosomal structural elements in E. coli RelA’s activation

 localize the (p)ppGpp-binding allosteric regulatory site of E. coli RelA

 test whether E. coli RelA can interact with uncharged tRNA off the ribosome

 determine the effect of antibiotics inhibiting translation on RelA-mediated

(p)ppGpp accumulation in biochemical system and as well as in live bacteria

Results and Discussion

Role of RelA and ribosomal structural elements in RelA activity (Paper I and II).

Recent cryo-EM studies of RelA have shed light on putative molecular mechanism of RelA-ribosome-tRNA interactions (80,89,91). According to the structures, RelA’s RRM and ZDF domains interact with A-site finger of the ribosome (ASF; helix 38), which plays an important role in positioning of the tRNA at the A-site (142,143), and S19 protein of 30S subunit (80). TGS domain of RelA interacts with acceptor (CCA) end of deacylated tRNA at the A-site, which infers the importance of TGS domain for RelA activation by tRNA (Figure 13).

Upon binding to ribosome in presence of RelA, the A-site tRNA adopts an unusual confirmation (A/R conformation), where it is distorted in a way that its acceptor stem is bent out from the peptidyl transfer site (Figure 13). In such conformation, tRNA elbow contacts sarcin-ricin loop (SRL) of the ribosome and RNA part of L7/L12 stalk of 50S subunit. These interactions may potentially stabilize tRNA in A/R conformation and contribute to RelA activation. In addition, interaction with thiostrepton loop (TL; a part of L7/L12 stalk), carrying antibiotic thiostrepton binding site (144), suggests a mechanism for RelA activity inhibition by the antibiotic.

Capitalizing on the structural findings, we test the role of both RelA (Paper I, unpublished data) and ribosomal structural elements (Paper II) in stringent response induction during amino acid starvation. Both of these studies aim to elucidate the same mechanism, however, they focus on different structural elements. To provide a more detailed picture of the mechanism of RelA’s activation on the ribosome it is reasonable to discuss the findings from these two papers together.

In Paper I, in order to test the role of C-terminal domains of RelA in its activity and allosteric regulation, we constructed 3 mutants: RelA-ΔRRM (amino acid residues 1-657), RelA-ΔZFD (amino acid residues 1-592), and RelA-ΔCTD (amino acid residues 1-385). In these mutants C-terminal domains were progressively deleted, so RelA-ΔRRM is missing RRM domain, RelA-ΔZFD is missing both RRM and ZFD domains, RelA-ΔCTD is missing RRM, ZFD, and TGS domains. In Paper II we employed ribosomes with mutations in structural elements of interest: a) ASF, where a part of helix 38 (20 or 34 nucleotides) was deleted, b) mutation in SRL (A2660C/G2661A) or SRL cut by sarcin, and c) mutation in TL (A1067U or Δ1067A).

We conducted our experiment in a biochemical system constituted from purified components such as vacant ribosomes or “starved” ribosomal complexes (SC) programmed with mRNA and carrying tRNA in the A-site, tag-less RelA (WT or mutants), and substrates (ATP+GDP/GTP). Mutation in ASF, as expected, affected activation by deacylated tRNA significantly (Paper II, Figure 4B). Surprisingly, shorter truncation (20 nucleotides) caused almost complete loss of activation both by vacant 70S ribosome and SC, whereas larger truncation (38 nucleotides) has mostly affected activation by SC. The activation of RelA by vacant ribosomes was affected only moderately. This points to the importance of ASF for accommodation of both RelA and tRNA.

The importance of the interaction between ASF and RelA is confirmed by our data with RelA ΔRRM and RelA ΔZFD construct (manuscript, Paper I). Deletion of RelA’s domain, which are in contact with ASF, leads to significant drop in RelA’s activity. RelA ΔRRM (amino acid residues 1-657) is 15 times less active in presence of SC and ppGpp and 10 times less active in presence of SC and pppGpp than the 744 amino acid residues long WT (Paper I, Figure 2). The picture is similar for the activation by vacant ribosome where RelA ΔRRM is 4 times less active in presence of ppGpp and 2 times less active in presence of pppGpp than the WT RelA. At the same time, there is still some residual effect of tRNA on RelA ΔRRM activation, which is completely gone in case of RelA ΔZFD (amino acid residues 1-592) where both ZFD and RRM domains are deleted. Besides no response to the presence of the deacylated tRNA, a weak ability of RelA ΔZFD to be activated by vacant 70S ribosomes is observed in the presence of pppGpp only (Paper I, Figure 2). Therefore, our data suggest that ribosomal ASF, as well as both RRM and ZFD domains, are required for proper accommodation of RelA on the ribosome and its activation.

A recent study on domain function of bifunctional RSH from Staphylococcus aureus (RelSau) employed similar approach and studied effects of truncations/mutations in CTD of RelSau and showed that TGS and ZFD motifs affect RelSau ribosome interaction the most, while ACT (RRM) motif appeared to be unimportant for the stringent response induction (145). It was also shown that RelSau CTD is not required for hydrolase activity and that the enzyme, which lacks it, is constantly in hydrolase ON/synthase OFF state in S. aureus background. However, in E. coli background the truncated protein was in hydrolase OFF/synthase ON state. This infers a substantial difference in the mechanism of synthase domain activation of E. coli and S. aureus RSHs.

Despite the fact that the interaction between A-site tRNA and SRL has been identified on cryo-EM structure, mutations in SRL as well as its cleavage by α- sarcin led to loss of RelA activation by vacant 70S ribosome. However, the alternations in SRL did not affect activation of RelA by deacylated tRNA of SC, indicating that SRL may take part in triggering spontaneous activity of RelA whithout playing a significant role in RelA activation by ribosome-tRNA complex, more specifically in A-site tRNA accommodation on the ribosome (Paper II, Figure 4B).

Mutation in thiostrepton loop (TL) appeared to be unessential for RelA activation. Deletion of A1067 or its mutation to A1067U did not cause profound effect on tRNA activation of RelA. However, A1067U mutation significantly reduced activation of RelA by vacant 70S ribosome (Paper II, Figure 4B).

It is quite surprising that TL and SRL, though interacting with A-site RNA in A/R conformation did not affect RelA activation by SC considerably. At the same time A1067 is crucial for binding of antibiotic thiostrepton (144), which completely inhibits tRNA dependent RelA activation. This suggests that A1067, though being important for thiostrepton binding, does not affect the role of thiostrepton loop in tRNA accommodation. Meanwhile, RelA’s activation by vacant ribosome is affected more profoundly by mutation/cleavage of SRL or mutation of TL.

As our preliminary results (Paper I, Figure 2) with RelA truncations suggest the activation by vacant ribosomes did not involve CTD and can be observed even for 385 amino acid residues long N-terminal part of the enzyme. This finding is in line with findings of Gratani and colleagues (145), which showed the same effect for truncated RelSau in E. coli background. This indicates a possible interaction between NTD, SRL and TL, which can trigger RelA’s activity. In case of full-length WT the activation by vacant 70S ribosome also involves ASF.

Allosteric regulation of RelA by (p)ppGpp (Paper I and II)

From previous studies it was shown that ppGpp exhibits ability to activate RelA allosterically if present in concentrations bellow 100 µM but inhibits RelA activity at higher concentrations (26,146). The effect of pppGpp has not been studied. The exact mechanism of this regulation is unclear and the position of the allosteric regulation site has not been established yet. However, it was proposed that this site may lie within the CTD (147). In our work, we investigated the effect of both products pppGpp and ppGpp on activity of E. coli WT RelA and truncated RelA in three different conditions: when RelA was a) present alone, b) in presence of 70S ribosomes and c) in presence of ribosomal “starved” complexes (SC).

It appeared that pppGpp and ppGpp have different effects on WT RelA activity depending on the substrate (Paper II, Figure 2D-F). pppGpp has a stimulatory effect on RelA activity regardless of the substrate. At the same time, ppGpp has stimulatory effect only when GDP was used as a substrate. In case of GTP, ppGpp inhibits its conversion.

These findings are in line with the fact that ppGpp has a stronger effect on E. coli transcription than pppGpp (38). The biological meaning of such phenomenon for the cell would be to produce the most efficient metabolic regulator as rapid as possible. Upon sensing amino acid deficiency, bacterial cell first starts to produce pppGpp from GTP, since it is the most abundant of guanosine nucleotides. The pppGpp produced stimulates conversion of GDP to ppGpp, in addition, GppA increases ppGpp pool by converting pppGpp to ppGpp (24).

The difference in effects between ppGpp and pppGpp is also observed in all our truncated constructs. pppGpp amplifies the effect of RelA truncations in all cases making them more pronounced. Addition of pppGpp increases enzyme’s activity and highlights the effect of a truncation. All truncated constructs in corresponding conditions show 3-5 fold higher activity in presence of pppGpp than in presence of ppGpp. Even 385 amino acid residues long RelA ΔCTD polypeptide shows increase in activity in presence of pppGpp, which can reach 8 fold difference in conditions where the protein is present without ribosomes (Paper I, Figure 2). These results suggest that, firstly, the presence of pppGpp makes RelA adopt a conformation that is different from the one in presence of ppGpp. Secondly, putative site for allosteric regulation of RelA lies within its NTD and not CTD as it was speculated before(148).

Role of RelA C-terminal domains in autoinhibition

In our studies, we also see the effect of RelA CTD domain in autoinhibition in accordance with the model proposed earlier (82,91,149,150). The model states that CTD prevents RelA from spontaneous activation through NTD-CTD cross- domain interaction and the formation of so-called “closed” conformation, alternatively RelA can form dimers through CTD-CTD protein interactions (86). In presence of SC (stringent response conditions) the activity of RelA ΔCTD is 12 times lower (in the presence of ppGpp) and 6.5 times lower (in presence of pppGpp) than WT RelA. Nevertheless, when the protein is present alone in the biochemical system, RelA ΔCTD activity in presence of pppGpp is twice higher than WT RelA. The deletion of C-terminal part of RelA prevents autoinhibition leading to its constant activation. This low but constant activity can contribute to reported growth defects of the strains expressing truncated versions of corresponding RSHs: RelSeq, (81), RelAEco (149,151), and RelMsm (152).

It is worth noting that the presence of TGS domain is already enough for the protein to be inert. Interestingly, we can see a weak, tRNA dependent difference in activation of RelA ΔCTD in presence of pppGpp (Paper I, Figure 2). This may indicate that pppGpp causes such conformational changes in RelA that it can interact with tRNA in the A-site. Most likely this interaction is different from the interaction with full length RelA since, according to cryo-EM structure, tRNA takes unusual A/R-conformation only when WT RelA is present.

The observation that RelA ΔCTD can be activated by vacant ribosome suggested that not all possible RelA-ribsome interaction were revealed by the structural studies. Though, it was shown that sarcin-ricin loop (SRL), thiostrepton loop (TL) and A-site finger of the ribosome are important for such interaction, the absence of the direct contact between RelA and these elements (except ASF) points to the fact that RelA can take a different conformation on the ribosome. This explains the activation of RelA truncates by vacant 70S ribosomes, with RelA ΔCTD being the most active.

RelA interaction with tRNA off the ribosome (Papers I and II)

Structural evidences prove tRNA-RelA interaction on the ribosome through RelA’s TGS domain (80,89,91) and indicate the possibility of tRNA-RelA complex formation in the cytosol, off the ribosome, as advocated by Winther and colleagues (153). According to them, RelA binds deacylated tRNA first and then the formed complex binds the ribosome, which leads to RelA activation. At the same time, RelA with mutated His432, which is interacting with CCA end of the tRNA (80), was not able to bind either tRNA or rRNA according to cross-link experiment (153). The question whether RelA can interact with tRNA off the ribosome has been asked before by Knutson-Jenvert et al. (87). Their filter- binding assay suggests that RelA does not interact with the tRNA in cytosol. Though, some tRNA was bound to the filter in the presence of RelA, authors found the specificity of such binding questionable (87).

Firstly, to detect binding of tRNA to RelA, we employed electrophoretic mobility- shift assay (EMSA). As it is seen from the gel (Paper II, Figure 5A), no interaction was detected even when RelA was present in 40 times molar excess. Considering the possibility that the RelA-tRNA interaction can be weak and not detectable by EMSA, we decided to probe it using RelA activity as a proxy. Secondly, we investigated if excess of non-cognate tRNA in the system with RelA and ribosomal “starved” complexes can inhibit RelA activity due to its sequestration. We titrated non-cognate tRNAVal in our biochemical system with SC and RelA as well as in the system where cognate tRNA was absent. In both cases, non-cognate tRNA does not cause significant drop in RelA activity (Paper II, Figure 5C)

If the proposed model of RelA interacting with tRNA off ribosome is true, then binding of tRNA to RelA may also disengage the mechanism of autoinhibition by changing conformation of C-terminal domain. We verified this hypothesis and found that titration of deacylated tRNA in the biochemical system in the presence of RelA alone does not affect ppGpp production (Paper II, Figure 5B).

We also tested (unpublished manuscript, Paper I) the effect of His432 mutation on RelA’s activity in our in vitro system. As shown in PaperI, Figure 4 , RelA His432Glu can not be activated by deacylated tRNA of “starved” ribosomal complex. This is in accordance with structural data (80) and cross-link experiments (153). However, RelA His432Glu still can be activated by the ribosome. This indicates that RelA His432Glu interacts with the ribosome but not deacylated tRNA.

Interestingly enough, His432 is also crucial for autoinhibition mechanism. When protein was alone in the biochemical assay RelA His432Glu was 5 times more active than the WT in presence of pppGpp and 2 times more active in presence of ppGpp.

Our data suggest that WT RelA as well as RelA His432Glu are capable of interacting with the ribosome. The excess of deacylated non-cognate tRNA does not sequester RelA and does not prevent its activation in our assay. These data point that, though, RelA may interact with deacylated tRNA off the ribosome, it is not a prerequisite for RelA’s binding to the ribosome. The effect of interaction between RelA and vacant ribosome is much more pronounced than the effect of interaction between RelA and tRNA. We can see that RelA activation by tRNA happens only upon its binding to A-site of the ribosome but not in the cytosol.

Effect of translational inhibitors on RelA induced stringent response (Paper III)

Increased level of (p)ppGpp is a cause of significant changes in cell metabolism and leads to increased virulence, bacterial survival and adaptation to unfavourable conditions and antibiotic tolerance (17,18). This and the fact that RSHs are absent in eukaryotes make them a perspective target for antimicrobial drug development.

In our study of RelA inhibitors we capitalize on the fact that (p)ppGpp production and protein synthesis is connected. It was shown previously that antibiotics which inhibit translation can cause decrease in (p)ppGpp accumulation presumably by increasing the pool of charged tRNA in the cell (154) or interfering with uncharged tRNA binding to the A-site (87,155).

In this paper we tested how thiostrepton, chloramphenicol and tetracyclin affect RelA activity in our biochemical system comprised of purified ribosomes, tRNA, RelA and nucleotides. Antibiotic thiostrepton (Figure 16) binds 50S ribosomal subunit between L11 protein and helices H43 and H44 of 23S rRNA. In this position thiostrepton is preventing binding of tRNA at the A-site (87) and GTPase activity of EF-G (156), impairing both polypeptide elongation and RelA activation by uncharged tRNA. The biggest drawback of thiostrepton as antibiotic, however, is its poor solubility in water, it is soluble in DMSO (157). For our experiments, we used non-ionic surfactant pluronic F-127 to facilitate thiostrepton (Paper III, Supplementary Figure 3).

As expected thiostrepton dramatically inhibits RelA activation by polyU programmed ribosomes in presence of deacylated tRNAPhe (Paper III, Figure 1D). At the same time, the activation of RelA by vacant ribosomes, as well by the ribosomes with a mutation in thiostrepton loop (A1067U), (Paper II, Figure 4B) which hinders thiostrepton binding to the ribosome, was not affected. This indicates high specificity of the mechanism of inhibition. The effect of the antibiotic can be observed at low concentrations. It abolishes RelA activation by deacylated tRNA already at o.5 μM. The same effect is seen for the biochemical system where ribosomal initiation complexes were assembled enzymatically by mixing ribosomes, mRNA (MF), tRNAPhe, tRNAifMet, and E. coli initiation factors (Paper III, Figure 20E and F). In this case, reaction was performed at 5 mM Mg2+, whereas the biochemical system with polyU programmed ribosomes contained 15 mM Mg2+.

Other antibiotics that we used in this study were tetracycline and chloramphenicol (Paper III, Figure 2&3). Tetracycline, is known to bind to A- site of the ribosome in several places preventing tRNA accommodation (155), while chloramphenicol is known to bind peptidyl transfer site of the ribosome inhibiting polypeptide chain elongation (154). In this way, chloramphenicol inhibits RelA indirectly by stalling translation, thereby, increasing the pool of charged tRNA in the cell.

In our experiments, tetracycline had moderate effect on tRNA dependent RelA activity, which was incresing with increase in antibiotic concentration. It is not completely clear, why tetracycline effect was not very pronounced, though it is supposed to effect RelA activation by deacylated A-site tRNA. One possible explantion can be that Mg2+ ion concentration was high (15 mM) allowing tRNA to compete with tetracycline for A-site.

Figure 16. Chemical structure of Thiostrepton

Thiostrepton is a oligopeptide antibiotic synthesized by some strains of Streptomyces. Upon binding to the ribosome it inhibits GTPase activity of EF-G and binding of A-site tRNA.

We also studied the effect of tetracycline, chloramphenicol and thiostrepton on stringent response in bacterial cultures. We grew E.coli (BW25113) and B. subtilis (BSB1) in MOPS minimal medium supplemented with 0.4% glucose and 20 amino acids. Unfortunately, E. coli culture is not affected by thiostrepton because of low uptake of the antibiotic (157). Experiments with thiostrepton were carried out only for B. subtilis culture.

To cause ppGpp accumulation, bacterial cultures were pretreated with antibiotic mupirocin at a concentration that inhibits the growth rate by 50% (70 μM in case of E. coli and 70 nM in case of B. subtilis). Mupirocin inhibits tRNAIle synthase causing Ile codon starvation during protein biosynhtesis. After mupirocin treatment, increasing concentrations of antibiotics suppressing protein biosynthesis were added. To observe the effect of the antibiotics, OD measurement of the cultures were taken and ppGpp in bacterial samples was quantified using HPLC method. In all cases translational antibiotic treatment led to a drop in growth rate and decrease in ppGpp level in E. coli (Paper III, Figure 2) and B. subtilis (Paper III, Figure 3). In contrast, control antibiotic trimethoprim, which inhibits dihydroftolate reductase causing inhibition of purine, methionine, and glycine biosynthesis (158), inhibits growth but does not affect ppGpp level.

We also observed that tetracycline, which inhibited ppGpp synthesis in our biochemical system only at high concentrations, caused significant decrease in ppGpp level in living bacteria at much lower concentrations.

Chloramphenicol also has much more pronounced effect on ppGpp synthesis in bacterial culture than in biochemical system. For chloramphenicol, it is in line with its mechanism of action. Inhibition of translation, due to obstruction of ribosomal peptidyl transfer site, leads to increase in charged tRNA pool and abolishes the stringent response.

Translational inhibitors showed to be efficient against the stringent response with thiostrepton being the most effective in case of B. subtilis. Despite its high efficiency, poor solubility in water (Paper III, Supplementary Figure 3A) make its use limited to ointment forms. Further structural modification is required in order to improve its solubility.

Conclusions

 A-site finger is crucial for RelA activation by ”starved” ribosomal

complexes and vacant 70S ribosomes

 RelA’s affinity to deacylated tRNA is low and the complex cannot be

detected by EMSA

 pppGpp is dramatically more potent activator of RelA than ppGpp

 (p)ppGpp allosteric regulation site is located in RelA’s NTD

 NTD of RelA can interact with and be activated by vacant ribosomes

 Translational inhibitors thiostrepton is a potent and a specific inhibitor

of RelA

 Antibiotics inhibiting translation universally abolish production of

(p)ppGpp in live cells

Acknowledgements

First and foremost, I would like to thank my supervisor Vasili Hauryliuk for opportunity to conduct my PhD studies and accomplish my work in his lab, and for his guidance and help during my studies.

I would like to express my gratitude to all the people involved in the process of my education: my co-supervisor Jörgen Johansson, examiner Sven Bergström, Gemma Atkinson, Anna Fahlgren, Victoria Shingler, Åke Forsberg, Ulrich von Pawel-Rammingen, and Mikael Wikström for their help and guidance.

Special thanks to Mikael Lindberg for taking care of all DNA constructs design and facilitating my work greatly.

I express my gratitude to all my colleagues and friends with whom I have been working these years: Villu and Marje Kasari, Yasuhiko Irie, Jelena Beljantseva, Liis Andresen, Steffi Jimy, Tõnu Margus. A special thank you to Roshani Payoe and Pavel Kudrin for sharing their knowledge and for their friendship, and to Hiraku Takada and Vallo Varik for their friendship and interesting discussions.

I would also like to acknowledge the administrative and technical staff of Molecular Biology Department for their help: Marek Wilczynski, Maria Westling, Patrik Holmquist, Johnny Stenman, and the Media Department staff without whom my work would be impossible.

I also would like to acknowledge my partners from Atack och Försvar. It was a pleasure to work with you.

Special thanks to Akbar Espaillat for being a helpful colleague and a great friend and to Naresh Chandra for being a great friend and a corridor mate.

My gratitude to all Charpentier lab and Cava lab members for being wonderful friends, lab and office neighbors.

My sincere gratitude to my dear Radha for her advice, encouragement and thorough proofreading of my thesis.

My sincere gratitude to my parents for their unconditional love and support.

Я дуже вдячний моїм батькам за підтримку усіх моїх зусиль і віру у мій вибір. Дякую за ваші любов, турботу і терпіння.

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