The ribosome, stringent factor and the bacterial stringent response

Rose-Marie Jenvert

Stockholm University

©Rose-Marie Jenvert, Stockholm 2007 ISBN 978-91-7155-414-7, pp 1-55 Printed in Sweden by Printers name, City 2007 Distributor: Department of Cell biology, The Wenner-Gren Institute, Stockholm University

To Niclas, �You�re my sun, my moon, my guiding star�

Abstract

The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.

List of papers

This thesis is based on the following studies, which will be referred to by their roman numerals in the text.

I. Rose-Marie Knutsson Jenvert and Lovisa Holmberg Schiavone (2005)

Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli. FEBS Journal 272, 685-695.

II. Rose-Marie Jenvert and Lovisa Holmberg Schiavone (2007)

The Flexible N-terminal Domain of Ribosomal Protein L11 from Es-cherichia coli Is Necessary for the Activation of Stringent Factor. JMB 365, 764-772.

III. Rose-Marie Jenvert and Lovisa Holmberg Schiavone (2007)

Mapping the interaction between stringent factor and the ribosome by footprinting of ribosomal RNA. Manuscript.

Additional publication (not included in the thesis):

Martin Högbom, Ruairi Collins, Susanne van den Berg, Rose-Marie Jenvert, Tobias Karlberg, Tetyana Kotenyova, Alex Flores, Gunilla B. Karlsson Hedestam and Lovisa Holmberg Schiavone (2007) Crystal Structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP. Submitted

Published papers were reproduced with permission from the publisher

Contents

Introduction ...............................................................................................11

The stringent response..............................................................................12 RelA and SpoT............................................................................................................14 (p)ppGpp.....................................................................................................................15

ppGpp interacts with the RNA polymerase ............................................................15

The stringent factor ...................................................................................17 The relA gene .............................................................................................................17 Secondary structure of SF...........................................................................................17

Crystal structure.....................................................................................................19

The ribosome ............................................................................................21 The structure of the prokaryotic ribosome ...................................................................22

The 30S subunit.....................................................................................................23 The 50S subunit.....................................................................................................25 tRNA binding sites .................................................................................................27 The GTPase associated region..............................................................................28 The peptidyl transferase centre..............................................................................29 Sarcin/ricin loop .....................................................................................................29

Dynamics of the ribosome...........................................................................................29

The ribosome and the stringent factor........................................................31 Ribosome-dependent activation of SF ........................................................................32 SF and ribosomal protein L11 .....................................................................................32

SF binds to the L11BD...........................................................................................32 L11 and the activation of SF ..................................................................................33

SF and the sarcin/ricin loop in the ribosome................................................................35 SF and the A-site on the ribosome..............................................................................36

The A-site and the activation of SF ........................................................................36 SF binds to domains involved in binding of tRNA...................................................37

Conclusion ................................................................................................38

Future perspectives...................................................................................39

Sammanfattning ........................................................................................40

Acknowledgement .....................................................................................42

References................................................................................................45

Abbreviations

SF stringent factor RNA ribonucleic acid rRNA ribosomal RNA tRNA transfer-RNA mRNA messenger-RNA ppGpp tetra guanosin phosphate pppGpp penta guanosine phosphate α2ββ´ RNA polymerase core σ sigma factor NTD N-terminal domain CTD C-terminal domain L11 ribosomal protein L11 L11BD L11 binding domain L10 ribosomal protein L10 L12 ribosomal protein L12 L7 ribosomal protein L7 A-site amino acyl-tRNA P-site peptidyl-tRNA E-site exit-tRNA GAR GTPase associated region EF-Tu elongation factor Tu EF-G elongation factor G IF-2 initiation factor 2 RF-3 release factor 3 PTC peptidyl transferase centre SRL sarcin/ricin loop ASL anticodon stem loop of tRNA CP central protuberance ASF A-site finger RIP ribosomal inactivating protein

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11

Introduction

�It is not the strongest of the species that survive, nor the most intelligent, but the one most responsive to changes� (Charles Darwin)

The ability to adapt to the environment is an important characteristic for

an organism and those that do not show such skills face the risk of being eliminated. Bacteria have during evolution developed many cellular regula-tory mechanisms to survive the different stress conditions that occur during their lifecycle. For instance lack of food and oxygen or the presence of excess salts or inhibitory agents, such as drugs, which affect normal bacterial growth. Most of these regulatory mechanisms occur at the genetic level and allow the bacteria to maintain a balanced and rather constant cellular compo-sition. A very important adaptive response in bacteria and one of the most studied is the stringent response.

Due to its role in growth and control of gene expression, the bacterial

stringent response has remained the subject of active interest over many years [1]. The initiation of the stringent response is the synthesis of a so called alarmone, (p)ppGpp. This alarmone of the stringent response controls several events in the life of a bacterium depending upon the environment in which the bacteria live, including sporulation, biofilm-formation, symbiosis and virulence (reviewed in [2]). In other words the stringent response modifies the physiology of the bacterium to such an extent that survival is permitted during the different environmental circumstances in their lifecycle.

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The stringent response

The stringent response in bacteria plays a significant role in the survival of bacteria under different environmental conditions (summarized in Figure 1). Repression of the transcription of stable RNA species, like tRNA and rRNA, and the up-regulation of transcription of genes coding the enzymes involved in amino acid biosynthesis are some of the effects during stringent conditions [3]. The stringent response is triggered during nutrient starvation by a protein called stringent factor (SF) [1]. SF binds to a stalled ribosome that contains an unacylated tRNA in the amino acyl-site (A-site) since there is a lack of amino acids [1, 4-7]. This leads to synthesis of an unusual guanosine phosphate, an alarmone called (p)ppGpp. The alarmone ppGpp interacts with RNA polymerase (RNAP), that is responsible for transcription of DNA to RNA in the cell and this results in a rapid down-regulation of stable RNA biosynthesis (tRNA and rRNA) and an up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis [1].

Figure 1: The stringent response is activated upon nutrient deprivation or stress in prokaryotes by the binding of stringent factor (SF, RelA) to translating but stalled programmed ribosomes with an unacylated tRNA in the ribosomal A-site. This leads to synthesis of the alarmones (p)ppGpp and binding of ppGpp to the RNA poly-merase.(Figure 28-24 in Principle of Biochemistry forth addition, Lehninger).

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Extensive research has been performed to reveal the functional significance of (p)ppGpp [8, 9]. The stringent response and ppGpp were found to play a significant role in the survival of bacteria under different environmental conditions and also to have a wide array of effects on the physiology of bacteria. It is also clearly shown that the response controlled by ppGpp is widespread and involves many features important for cell physiology not only during growth but also during stationary conditions [2].

In some bacteria, such as Bacillus and Clostridium spp. the response to starvation is to form metabolically inactive endospores which are highly resistant to starvation and stress such as heat and dehydration [2]. The stringent pathways have also been found to be important for the virulent attributes that help pathogen survival in the host. In Mycobacterium tubercu-losis the stringent response was found to important for their ability to survive under prolonged starvation and anaerobic conditions, an environment possibly encountered by bacteria inside the host [10, 11]. One of the physiological effects that might play a part in the survival of M. tuberculosis during these conditions is the morphology, since an altered morphology was observed in the absence of the stringent response in M. smegmatis an often used model organism for M. tuberculosis [12, 13], see Figure 2.

Figure 2: Effects of stringent response upon the appearance of M. smegmatis. Colonies were grown for 3 days in 7H9 liquid medium (1) Wild-type M. smegmatis and (2) M. smegmatis ∆relMsm. The lack of stringent response altered the cell mor-phology and the cells appeared significantly longer cells than the wild-type cells [13].

Another physiological effect that might play a part in the survival of M. tuberculosis could is the ability to adhere to the host surface. This is also an important property for the virulence of a pathogen, which in turn produces a biofilm and ppGpp has been reported to play a crucial role in this biofilm formation [14]. ppGpp was also shown to be required for the expression of nearly all known virulence genes in Salmonella in response to

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growth conditions relevant to host infection and required for regulation of both the extracellular (invasion) and intracellular virulence genes [15]. The involvement of stringent factor in the virulence and pathogenesis in bacteria, illustrated above makes it important to elucidate the mechanisms for the stringent response and to find out if some or all of the properties important for the survival of pathogenic organisms can be stopped by inhibiting the stringent response. This possibility to stop the growth of pathogens is even more important in the light of that many pathogens today are becoming resistant to antibiotics.

The stringent response has been suggested to be present not only in

bacteria but also in plants. A functional homologue of relA/spoT was found in Arabidopsis thaliana [16] and the presence of ppGpp and bacterial type RNAP in chloroplasts [16, 17] strengthened the idea of a stringent response in plants. When plants were treated with plant hormones known to play a critical role in signal transduction [17] an increased amount of ppGpp was synthesized and the homologue of relA/spoT was suggested to interact with the plant defense system [16]. It was therefore suggested that the stringent response is involved in cell signaling in plants, as a response to environmental fluctuations, including salt, UV radiation, acid and alkali, pathogen infection and drought etc.[16, 17].

RelA and SpoT In Escherichia coli and several other Gram-negative bacteria the stringent

response is regulated by two genes, relA and spoT [18]. relA was first called the RNA control gene because the first discovered response was rapid accumulation of stable RNA (rRNA and tRNA) [18]. There are two different pathways responsible for maintaining the levels of (p)ppGpp in the cell, the RelA and SpoT-dependent pathways. The RelA-dependent pathway involves the protein from the relA gene, RelA or SF which is associated with ribosomes and responsible for the (p)ppGpp synthesis during nutrient starvation [1], and will be discussed in more detail later. The SpoT-dependent pathway involves the protein from the spoT gene, SpoT which displays (p)ppGpp hydrolase activities and is responsible for degradation of (p)ppGpp in the cell but has also a low synthetic activity [19]. Little is known about how SpoT senses starvation conditions and the mechanism(s) behind SpoT-dependent production of ppGpp [20].

In Gram-positive bacteria, a single bifunctional protein, Rel is responsible

for the regulation of (p)ppGpp in the cell and carries out both synthesis and hydrolysis [21, 22]. The crystal structure of the catalytic domain of Rel has been solved and will be described later. The presence of the single

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bifunctional enzyme was however found to be restricted not only to Gram-positive bacteria, but has recently also been demonstrated in Gram-negative bacteria [23, 24].

(p)ppGpp The alarmones implicated in the stringent response were initially called

the �magic spots� MSI and MSII [18] and were later identified as guanosine tetra phosphate (ppGpp) and guanosine penta phosphate (pppGpp), collectively called (p)ppGpp [25]. The synthesis of (p)ppGpp during the stringent response is triggered by the activation of SF, which leads to donation of β- and γ-phosphates from ATP to GTP or GDP, the major product in vivo being pppGpp. The degradation of pppGpp to ppGpp is catalyzed by (p)ppGpp 5´phosphorylase (GPP) and then ppGpp acts as an effector molecule by binding to RNAP (see Figure 1). SpoT is responsible for the degradation of ppGpp in the cell into GDP or pppGpp to GTP but was also suggested to have a low synthetic activity [1]. The (p)ppGpp metabolism is summarized in Figure 3.

Figure 3: Cellular routes of (p)ppGpp metabolism. The enzymes responsible for the different steps of the (p)ppGpp metabolism are represented by their genes. relA, spoT, gpp and ndk (nucleoside 5´diphosphate kinase).

ppGpp interacts with the RNA polymerase The alarmone ppGpp was shown to interact with the RNAP in the cell.

The RNAP consists of two α-subunits and two β-subunits, β and β´ that together form the RNAP core (α2ββ´). Sigma (σ) factors (in E. coli, seven different) can associate with the core enzyme and give specificity for a particular promoter and transcription of specific genes. ppGpp was suggested to interact with the β and β´ subunits of the RNAP core enzyme [26-28] and this was later confirmed in the structure of RNAP co-

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crystallized with ppGpp where ppGpp was seen to interact with both subunits near the active site of RNAP [29].

Effects of ppGpp on RNAP promoter interaction When ppGpp is bound to RNAP it can act both as a positive and a

negative regulator of transcription. In general, σ70-dependent genes involved in cell proliferation and growth are negatively regulated by ppGpp [30-35], whereas the σ70-dependent genes involved in maintenance and stress defense in the cell are positively regulated by the alarmone [20, 36].

ppGpp has also been suggested to play a role in σ factor competition by

regulating the different binding abilities of σ factors to core RNAP [37, 38]. The sigma factor competition model suggests that during the stringent response, a shift in the ability of the sigma factors to recruit RNAP core enzyme could reduce the amount of RNAP core with bound σ70 in the cell. This then leads to reduced levels of the holoenzyme, mimicking an artificial underproduction of σ70 [39].

Recently it was shown that the effects of ppGpp on transcription are

increased by the presence of the protein DksA (a transcription factor) [40]. DksA, known to bind to the RNAP is suggested to increase the negative effects of ppGpp on rRNA promoters and also contribute to the positive effects of ppGpp [40] however the understanding of the mechanism behind the action of DksA is not clear and it is possible that ppGpp in some instances acts alone and in others in concert with DksA [20].

More research is needed to shed light in to the mechanisms behind to

effects of ppGpp during the stringent response.

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The stringent factor

In this thesis I will focus on describing SF and its role in the stringent response.

The relA gene The gene encoding SF in Gram-negative bacteria such as E. coli is the

relA gene. The relA gene was discovered already in 1961 by Stent et al [41] and was later mapped at 59.2 min on the E. coli chromosome [42]. The initiation of transcription from the relA gene can occur at two promoters, relAP1 and relAP2 that are suggested to be σ70-dependent [43]. The first promoter for relA in E. coli, relAP1 was found approximately 178 bp up-stream of the relA transcriptional start site [44] and the second promoter, recently found, is located 626 bp upstream [43]. The promoter relAP1 was suggested to be active during all growth phases and the transcription of relAP1 is dependent on an up stream-element (located about 40 bp upstream of the transcription start site) which makes the transcription more efficient [43]. The promoter relAP2 is thought to be transiently induced at the transi-tion state between the exponential growth phase and the stationary phase and transcription using this promoter seems to be regulated by globular factors [43].

The relA and spoT genes of Gram-negative bacteria have been suggested

to have evolved separately from an originally duplicated rel gene [45]. An amino acid sequence alignment of Rel, SF and SpoT show that the bifunctional Rel protein from a Gram-positive bacteria is more similar to SpoT of E. coli than SF, and this suggests that the bifunctional Rel is related to SpoT that also have both synthetic and hydrolysis activities [2].

Secondary structure of SF SF consists of 744 amino acids with a molecular mass of 84 kDa [44].

Physically and functionally, SF from E. coli can be divided into an N-terminal (NTD) and a C-terminal domain (CTD). The NTD is composed of amino acids 1-455 and the CTD of amino acids 455-744. The structure of

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SF is found to be mostly α-helical in nature as noted from CD experiments and also from the estimation of the secondary structure by PSIPRED, shown in Figure 3.

Figure 3: Secondary structure of SF as predicted by PSIPRED [46, 47].

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The NTD was suggested to contain the catalytic domain of SF and the CTD to be the ribosomal and tRNA binding domain responsible for activating the catalytic NTD. The structure of the CTD was suggested to be at least partially unstructured until binding to ribosome (amino acids, nucleo-tides, or uncharged tRNA) which would lead to the activation of the protein [48]. It was also suggested that the fusion of a structured NTD with unstructured CTD alters the overall conformation of the protein [48]. The idea that the CTD is responsible for regulating the activity of SF was suggested by a study showing that deletion of the CTD made the protein unresponsive to ribosome, mRNA and tRNA [49] and the CTD on its own was shown to have no synthetic or hydrolytic activity, thus restricting the catalytic activity to the NTD [48]. The CTD was also suggested to be involved in oligomerization of SF which could be a way of regulating the activity of the protein [50].

A mutation of a cysteine in E. coli SF, C638 resulted in a relaxed

stringent response i.e. the cells are deficient in the synthesis of pppGpp [2, 48, 50]. It was therefore suggested that this mutation in the CTD makes SF unresponsive to uncharged tRNA and that the cysteine is required for interaction with uncharged tRNA [2].

Further mutation studies must be done in order to learn more about which parts of SF that are important for the different functions of this protein.

Crystal structure Nearly three decades after the discovery of stringent response, the first

crystal structure of the NTD of the bifunctional protein Rel from a Gram-positive bacteria, Streptococcus equisimilis was published (shown in Figure 4) [51]. The NTD were found in two different conformations (Figure 4A and B) and show two catalytic domains in the NTD, the hydrolase domain and the synthetase domain that correspond to the two activities of RelSeq. In the structure the hydrolase domain (5-159) (Figure 4, green) and the synthetase domain (176-371) (Figure 4, yellow) are interconnected by a 3-helix bundle (Figure 4, red) [51]. The two different conformations of Rel were suggested to be responsible for switching on and off the two different activities of Rel and Hogg et al have suggested a model for regulating the two antagonistic activities [51]. A ��intramolecular cross-talk��, an interaction between the NTD and the CTD has been proposed to be necessary for regulation of the two antagonistic activities in the NTD [49] and the molecular structure of the NTD of RelSeq [51] further supports the two-domain cross-talk model for the synthesis and hydrolysis of (p)ppGpp. This self-regulatory mechanism due to structural changes in the protein might in the future be used to design inhibitors that specifically interfere with the active sites and these might have the potential to be developed into antibacterial drugs.

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Figure 4: Crystal structure of RelA from Streptococcus equisimilis, RelSeq 1-385. [51] Showing the two different conformations found when crystallised (A and B). The hydrolase domain (5-159) is shown in green, the synthetase domain (176-371) is shown in yellow and the 3-helix bundle is shown in red. The primary and secon-dary structure of RelSeq 1-385 is shown in C, with colors according to A and B. Reprinted from [51] with permission from Elsevier.

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The ribosome

Ribosomes are large ribonucleoprotein complexes, incredible molecular machines composed of both RNA and protein, which are responsible for the protein synthesis in all cells. Unlike other cellular polymerases, the mechanism of ribosomal action appears to be based fundamentally on RNA and today we know that the ribosome in fact is a ribozyme.

A translating ribosome consists of two subunits, the small subunit (30S

subunit) and the large subunit (50S subunit). The 30S subunit consists of 16S rRNA and 21 proteins whereas the 50S subunit consists of 23S rRNA, 5S rRNA and 31 proteins. Together these subunits form the 70S ribosome [52]. The cavity between the two subunits where tRNAs and translation factors bind is called the interface cavity. The two subunits of the ribosome have different functions during the protein synthesis. The 30S subunit contains the decoding region and is responsible for the binding to the Shine-Dalgarno sequence the mRNA and the interaction with the anticodon stem-loops of the tRNAs in A-, P- and E-site. The 50S subunit contains the peptidyl transferase centre (PTC), located in the 23S rRNA [53-55] that catalyzes peptide bond formation between the incoming amino acid on the A-site tRNA and the nascent peptide chain attached to the P-site tRNA, resulting in a growing polypeptide chain [53]. During translation, elongation factors and release factors interacts with the GTPase associated region (GAR) in the 50S subunit [56-61]. The GAR consists of the ribosomal protein L11 (L11) and its L11 binding domain (L11BD) in 23S rRNA, and their functions will be discussed more later on in the thesis. The 50S subunit also contains binding sites for tRNA, the A-, P- and E-site. The important features of the 30S and 50S subunits are shown in Figure 5.

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Figure 5: Crystal Structures of the 50S (left) and 30S (right) subunit of the 70S ribosome, showing the locations of the L7/L12 stalk, the L1 stalk, the A-, P-, and E-site tRNAs, the decoding site, the peptidyl transferase centre (PTC), the GTPase associating region (GAR) and the mRNA. Reprinted from [62] with permission from Elsevier.

The structure of the prokaryotic ribosome A major breakthrough for our understanding of the ribosome was

achieved some years ago when high resolution structures of the 50S and 30S ribosomal subunits were solved [54, 63]. Progress has also been made in obtaining structural data on the whole ribosome and recently, the structure of the E. coli ribosome was solved at 3.5 Šresolution [55], Figure 6. The complete structure of the E. coli ribosome is in my opinion the most important contribution to the ribosome structures since E. coli often is used as a model organism and also is the bacterium we have used in our studies of the stringent factor. The ribosomal structure from Schuwirth et al represent a detailed view of the complete ribosome and of the interface between the 30S and 50S ribosomal subunits, and also the conformation of the peptidyltrans-ferase center in the context of the intact ribosome as well as the domains of L1 and L11 and this gave us new information about the structural basis of ribosome dynamics [55] and the overall structure of the ribosome from this study is shown both in the ��standard�� view (A) and in a ��top�� view (B) in Figure 6.

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Figure 6: The structure of the complete ribosome 70S from Escherichia coli. (A) View from the solvent side of the 30S subunit. rRNA and proteins in the 30S subunit are colored light blue and dark blue, respectively. 23S rRNA and proteins in the 50S subunit are colored gray and magenta, respectively. 5S rRNA is colored purple. (B) View rotated 90- about the horizontal axis in (A). Letters indicate the approximate alignments of the aminoacyl (A), peptidyl (P), and exit (E) tRNA bind-ing sites at the subunit interface. The 5´ to 3´ direction of mRNA, which threads around the neck region of the 30Ssubunit, is also indicated. From [55] Reprinted with the permission from AAAS.

In the 30S subunit the visible features includes the head, body, shoulder, platform, and spur and in the 50S subunit, the main features include the L1 arm, consisting of ribosomal protein L1 (L1) and its 23S rRNA binding site, the central protuberance (CP), the RNA helix of the A-site finger (ASF) and the region near proteins L7/L12, which includes the L11 arm (consisting of protein L11 and its 23S rRNA binding site) [54, 55, 64-66]. Unfortunately the approximate location of some structural elements is not fully modeled because of disorder and this includes ribosomal proteins L7/L12 (L7/L12) and the tip of the ASF (shown in gray in Figure 6) that are known to be highly mobile within the ribosome [67-69]. The high resolution crystal structures are giving us a detailed view of how the ribosome is folded and are in agreement with previous footprinting and crosslinking studies on the ribosome [70, 71, 72]

The 30S subunit Several important features of the ribosome have been confirmed to be

located at the 30S subunit such as the mRNA channel [73] and the decoding centre [63, 74], shown in Figure 5. The 30S subunit is responsible for interacting with the mRNA during initiation and with tRNA during translation. These interactions are mainly done by the 16S rRNA of the 30S subunit, and the 16S rRNA can be divided into different domains: 5´domain,

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central domain, 3´major domain and 3´minor domain as shown in the secondary structure in Figure 7A. The tertiary structure of the 16S rRNA shown in Figure 7B is colored in the same way as the secondary structure and suggests a view of how the rRNA is folded into its proper structure. As seen in Figure 7B the 30S subunit consists of an elongated structure with a slightly bend head that is connected to the body via the platform [55, 63, 75]. Additional features of the 30S subunit are the neck, spur and shoulder. By comparing the colors in the seconday structure with the tertiary structure it shows that the body of the 30S subunit is formed mostly by the 16S rRNA 5´domain with the 3´minor domain in the centre. The 3´major domain of 16S rRNA forms the head and the central domain forms the platform of the 30S subunit.

Figure 7: The ribosomal RNA secondary structure (from Thermus Thermopilus). A. The 16S rRNA of the 30S subunit showing the 5´domain (5´), central domain (C), 3´major domain (3´M) and 3´minor domain (3´m). B. The tertiary structure of the 30S subunit with the rRNA colored as shown in A. From [75]. Reprinted with the permission from AAAS. [75]

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It has been suggested that the binding of tRNA to the A- and P-site or P- and E-site rotates the head of the 30S subunit about 6 º towards the E-site of the ribosome [55, 75, 76] and that this is a mechanism for controlling mRNA and tRNA movement during translocation [55]. This movement could also be a part of the �unlocking� step on the small subunit that is suggested to be required for mRNA and tRNA translocation [77-79].

The 50S subunit The 50S subunit contains the peptidyl transferase centre that catalyses the

formation of the peptidyl bond during the elongation step in translation. The flexible lateral stalk, the GAR and the L7/L12 stalk is other prominent features of the 50S subunit, which are important for interactions with factors and is discussed more later on in this thesis. The other flexible stalk, the L1 stalk is suggested to be important for release of E-site bound tRNA.

The 23S rRNA of the 50S subunits can be divided into different domains:

domain I-III (5´half), and domain IV-VI (3´half) as shown in Figure 8A. The tertiary structure of the 23S rRNA shown in Figure 8B is colored in the same way as the secondary structure and suggests a view of how the rRNA is folded into its proper structure. As seen in Figure 8B the 50S subunit has a hemispheric shape and the interface side, facing the 30S subunit is shown. By comparing the colors in the secondary structure with the tertiary structure it shows that the central protuberance at the top consists mainly of 5S and the center of domain V of 23S rRNA. The top of domain II of 23S rRNA together with the L7/L12 stalk and the ribosomal protein L11 is located on the right side of the structure [54]. The left side consists of part of the do-main V and the L1 stalk. The 50S subunit interface consists mostly of do-main IV of 23S rRNA [54, 75]. The ridge of the interface is composed of helix 67-71 in domain IV where helix 69 is responsible for interacting with the 16S rRNA in the 30S subunit [65]. The center of the subunit is made up by the central ring of domain V part of which is responsible for the peptidyl transferase activity of the ribosome [53]. Domain V also forms the binding site for ribosomal protein L1 and helps to stabilize the elongation factor binding part of the region [54]. The sarcin/ricin loop (SRL) located in do-main VI forms one lobe of the base of L7/L12 stalk where the other one is L11 and its RNA binding domain [54].

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Figure 8: A. The ribosomal RNA secondary structure (from Thermus Thermopilus). The 23S rRNA and 5S rRNA of the 50S subunit showing domain I, II, III, IV, V and VI of 23S rRNA. B. The tertiary structure of the 50S subunit with the rRNA colored as shown in A. From [75]. Reprinted with the permission from AAAS.

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tRNA binding sites Transfer-RNAs (tRNAs) are responsible for the decoding of the mRNA during translation and the anticodon loop in tRNA is responsible for the recognition of a specific codon on the mRNA. The crystal structure of tRNA is presented in Figure 9.

Figure 9: tRNA from yeast showing the acceptor stem, CCA-tail, anticodon loop and the D-loop [80].

There are three binding sites for tRNA on the ribosome, shown in Figure 5. An A-site, to which aminoacyl tRNAs are delivered in an mRNA-directed fashion, a P-site where peptidyl tRNAs reside, and an E-site through which deacylated tRNAs pass as they are released from the ribosome [81]. During translation the tRNAs move from the A-site close to the L7/L12 stalk base and along the intersubunit cavity via the P-site, towards E-site and the L1 stalk [65].

The tRNAs can be bound in classical states (A/A, P/P and E/E) or in

hybrid states [75, 82]. In the hybrid states model of translocation tRNA adopts hybrid configurations like A/P-P/E since the two ends of the tRNA move independently of each other on the ribosome [83-85]. Recently additional intermediate states have been described (see for example [86]).

In the crystal structure the A-, P- and E-site on the ribosome was shown

to cross the intersubunit cavity of the ribosome and are dominated by rRNA interactions [54, 87, 88]. The interactions with rRNA helps to stabilize the binding of tRNA to the ribosome but are also involved directly in the

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functional process and the ribosome contacts tRNAs at conserved parts of their structure [75].

Looking closer at each of the binding sites of the tRNA on the ribosome,

the A-site of the ribosome was found to be located in the decoding site of the 30S subunit where codon-anticodon interactions are monitored [82, 89]. At the 50S subunit the A-site is located within the PTC were the new amino acid at the 3´ end of tRNA is made available for peptide bond formation [53, 72, 87, 90] also the A-loop [91, 92] and the A-site finger [75] interact with the A-site bound tRNA. In the P-site the tRNA is held tightly in its position to maintain the reading frame and for peptidyl transfer [53, 87 90], this is done mainly through extensive contacts with both ribosomal subunits [65]. The anticodon stem-loop (ASL) of the P-site tRNA is in close contact with the cleft of the 30S subunit [87] and in the 50S subunit the P-site tRNA is in contact with the P-loop [82], and helix 69 in domain IV [88]. Unlike both the A- and P-site that are composed mainly of rRNA, the E-site is composed of both ribosomal proteins and rRNA. The ASL of the tRNA in the E-site is suggested to be located between the head and platform of the 30S subunit as seen in the crystal structure by Yusupov et al [75]. The L1 stalk in the 50S subunit binds to the E-site tRNA and the tRNA ASL makes a minor groove interaction with helix 68 [75]. The CCA tail of the E-site tRNA is found to directly contact C2394 of the 23S rRNA [93], and this interaction was earlier on suggested by chemical modification studies [82, 94].

The GTPase associated region The region of the prokaryotic 50S ribosomal subunit associated with

interactions of ribosome-dependent GTPase proteins such as initiation factors and elongation factors is referred to as the GAR. It has long been known that the GAR in bacterial ribosomes is involved in regulating several ribosomal factor-dependent processes, including the binding of EF-Tu and EF-G followed by translocation [95-97], initiation by initiation factor 2 (IF2) [59-61], termination by release factor (RF3) [98] as well as SF dependent synthesis of (p)ppGpp is dependent on this region [1, 6, 99-101]. The GAR has been extensively characterized by different methods including genetic, cross-linking, and chemical footprinting approaches, and it consists of the ribosomal protein L11 and its binding site on 23S rRNA [66] which is in close contact with the binding site of the pentameric complex forming the L7/L12 stalk (L10-(L7/L12)2) on the right shoulder of the 50S subunit [102, 103] and also with the highly conserved SRL domain [104].

29

The peptidyl transferase centre The ribosomal PTC catalyzes the two principal chemical reactions of

protein synthesis, the peptide bond formation and the peptide release. The primary function of the PTC is to catalyze peptide bond formation, adding the amino acids to the growing polypeptide chain on the tRNA. The second principle chemical reaction is peptidyl-tRNA hydrolysis, which is required for the termination of translation and release of the fully assembled poly- peptide from the ribosome. The active site of the PTC and its enzymatic activity of peptidyl transferase is suggested to be only dependent on rRNA because there are no ribosomal proteins closer to the reactive centre of a transition state intermediate than 18 Å [64]. All the bases involved in the peptidyl transferase reactions are located in the central ring of domain V within the single stranded regions and these are also closest to the catalytic site [64]. It is has been suggested that the base A2451 is essential for the catalytic reaction but the mechanism is still unclear [105-107] and the base A2602 is suggested to be involved in the peptide release [108, 109].

Sarcin/ricin loop The helix of the SRL is a highly conserved part of the ribosome (nucleotides 2646� 2674 in Escherichia coli), located below the L7/L12 stalk and is involved in the binding the elongation factors, EF-Tu and EF-G [110, 111] and IF-2 [112] and also a target of the ribosomal inactivating protein (RIP) α-sarcin [113, 114]. It has been suggested that the flexibility of the SRL facilitates the conformational changes during the elongation cycle giving the SRL an active role in elongation [113]. It is also possible that the only function of SRL during elongation is to bind EF-Tu and EF-G [110].

Dynamics of the ribosome Cryo-EM is a useful tool when studying the dynamics of the ribosome,

giving us a clue of how this molecular machine works and making it possible for us to understand the structure of the ribosome not only at high resolution but in different complexes representing many different states [115-118]. This will hopefully together with structure probing and crystal structures lead to a complete understanding of the ribosome structure and most importantly of functional ribosomal complexes in their true biological context. Functional ribosomal complexes analyzed by cryo-EM have already provided information on the structure of the ribosome trapped in different states of the translational process [67, 116, 119-122]. These structures indicates a large-scale molecular movement of the two subunits during movement of tRNAs on the ribosome [84, 119] and the involvement of L11 as a possible molecu-

30

lar switch during the elongation cycle [117]. Cryo-EM studies have also demonstrated among other findings, that all the involved factors during crucial steps of elongation, release, and recycling bind in the same funnel-shaped region in the interface cavity on the side of the L7/L12 stalk base. One of the binding sites for these factors was shown to be the GAR of the large subunit [84, 123]. The interesting thing about the structures obtained from cryo-EM described above is that they have not only revealed the 3D structure of the rRNA, ribosomal proteins and their interactions but have also lead to proposals for a mechanism of the protein synthesis.

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The ribosome and the stringent factor

The ribosome-dependence of SF has been known since the factor was first isolated more than 30 years ago but still there is more to be learned about how SF interacts with the ribosome. Due to the very low amounts of SF present in the cell [124, 125] it was hard to purify the protein but during the last few years several different recombinant pyrophosphoryl transferases have been cloned and isolated (paper I, [6, 22]). We were, as described in paper I able to obtain a concentrated recombinant protein of high purity and activity by using the inherent property of SF to precipitate at low salt con-centration. Studies of the recombinant SF have increased our knowledge and led us closer to reveal the secret behind the activation of SF and the stringent response but there is still more to discover about this interesting response in bacteria. For instance, how is SF bound to the ribosome and what parts of the ribosome are involved in the stimulation of (p)ppGpp-syntheisis by acti-vation of SF? The 50S subunit of the ribosome has been suggested to be involved in both the binding [126, 127] and activation of SF, and it has been suggested that the GAR, the L7/12 stalk, and also ribosomal protein L10 (L10) [128] are possible interaction sites for SF (paper II, [1, 6, 99, 100]). The need for an unacylated tRNA in A-site of the ribosome for activation of SF implicates that also the A-site or at least the A-site bound tRNA is involved in the binding of SF. However, the binding of SF to the ribosome is not dependent on the unacylated tRNA in the A-site [6] and this suggests the A-site on the ribosome to be a possible site for regulating the activation of SF.

The aims of this work have been to clone and purify the recombinant SF

(paper I), to learn more about SF and get a better understanding of how SF is activated by the ribosome by studies in an in vitro system. The importance of the ribosome, tRNA (paper I) and L11 (paper II) in the activation of SF was investigated and also how SF binds to the ribosome (paper III). In our recent study in paper III we studied the interaction between the ribosome and SF by chemical modification and subsequent primer extension analysis i.e. foot-printing and we found that SF footprints several important nucleotides of the ribosome, which will be discussed below together with the properties of the activation of SF. Here it should be mentioned that the footprinting method can not distinguish between direct interaction sites and conformational changes. However the interaction of several translation factors and tRNA

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with the ribsome has been mapped by footprinting and later on confirmed by other methods [75, 98, 110, 119]

Ribosome-dependent activation of SF Analysis of the purified recombinant SF in an in vitro system (described

in paper I) confirmed that the activity of SF is dependent on ribosomes, and that a template bound to the ribosome is needed for both the binding of SF to the ribosome and for activation of SF (paper I, [6]). The activation of SF and the production of (p)ppGpp was also found to increase with increasing amounts of ribosomes present, thus more ribosomes led to increased synthesis of (p)ppGpp until an optimum was reached when ribosomes were in 10-fold excess over SF (paper I, [6]). To try and explain the biological significance of this observation Wendrich et al suggested that SF can hop between different stalled ribosomal complexes and initiate pppGpp synthesis [6]. However I find it more likely that SF already is prebound to some ribosomes and when an unacylated tRNA binds in the A-site and/or a conformational change occurs in L11 (paper II, and below) which leads to the activation of SF.

SF and ribosomal protein L11 As mentioned earlier L11 was found to be involved in the activation of SF

since ribosomes that lack L11 are impaired in this function (paper II, [1, 6, 99, 100]). L11 is however not essential for the binding of SF [6] but SF probably binds close to the L11 on the ribosome since L11 is involved in the activation of SF. We have suggested that SF could bind to the 50S subunit of the ribosome in the vicinity of L11 (paper II). In paper II and paper III we look closer at this interesting association between SF and L11 and this is discussed in more detail below.

SF binds to the L11BD The interaction between SF and the ribosome was investigated by

chemical modification of ribosomal complexes with bound SF (paper III). The binding of SF to the ribosome revealed protected bases within the L11BD in domain II of the 50S subunit in close contact with the L7/L12 stalk (Figure 10, blue). It was interesting to finally be able to confirm that SF interacts with this region of the ribosome which have been known to be involved in the activation pppGpp-synthesis for long (paper I, [1, 6, 99, 100]). When analyzing the resulting protected phosphate backbones in the

33

L11BD when SF was bound to the ribosome and comparing it to the binding site of L11 we surprisingly found that the protected bases of SF and L11 do not overlap, shown in Figure 6 in paper III. This is interesting because it could explain why L11 is not essential for binding SF to the ribosome [6] but still essential for the activation of SF (paper II, [1, 99, 100]).

Figure 10: Mapping the SF footprints into the molecular model of the E.coli ribo-some (2AWB) [55]. Domain II, helix 42-44 (blue), domain V, helix 89 (red) and domain VI, helix 95 (SRL) (magenta), helix 92 also called A-loop (green) and helix 93 (pink).

L11 and the activation of SF As discussed above L11 and SF both binds to the L11BD of the 50S sub-

unit of the ribosome but their binding sites do not overlap. L11 is however essential for activation of SF and seems to take part in the regulation of SF and hence the (p)ppGpp-synthesis. How is then L11 involved in this regula-tion? L11 is probably not involved in the binding of SF to the ribosome since they do not share binding sites and this suggests that L11 on its own and the inherent properties within the protein are involved in the regulation of (p)ppGpp-synthesis. To try to answer the question of how L11 is involved in this regulation we designed mutants/deletions of L11 and analyzed their ability to activate (p)ppGpp-synthesis (paper II). L11 consists of a CTD and a NTD that is connected by a flexible linker region (Figure 11). The

34

L11CTD is responsible for the binding of L11 to the L11BD in the ribosome and the L11NTD is suggested to be flexible and is located on the surface of the structure when bound to the ribosome [66, 129].

Figure 11: The ribosomal protein L11. A. L11 is consists of an NTD and a CTD connected by a flexible linker. B. Crystal structure of L11 [129].

When ribosomes lacking L11 were reconstituted with the mutant or wild type L11 and tested for their ability to activate the synthesis of pppGpp by SF, we found that the L11CTD on is own is impaired thus confirming that the L11NTD that is involved in the activation (paper II). This was no surprise since a part of the L11NTD, the proline-rich helix already was shown to be involved in the activation of SF [100], later confirmed by us in paper II. The flexibility of the L11NTD is suggested to depend on the linker between the two domains in the protein [130, 131] and since it is possible that this flexibility is important for its regulatory role in (p)ppGpp-synthesis we made a mutation/deletion of this region which confirmed that the linker and thus the flexibility of the L11NTD is important for L11´s regulatory role in (p)ppGpp-syntesis. The NTD of L11 has also been suggested to act as a molecular spring in translation [117]. Looking closer at the structure of L11NTD the proline-rich helix located on the surface on the protein when bound to the ribosome [66, 129] could be a possible interaction site with SF since prolines are often found in close contact at protein-protein interactions sites [132]. The deletion of the entire proline-rich helix confirmed its

35

importance in the activation of SF and also suggested that the helix might be involved in a more general function on the ribosome besides the activation of SF (paper II) It is possible that the deletion of the proline-rich helix resulted in a distortion of the structure and this could affect the result but a mutation in the proline-rich helix of L11, proline 22 confirmed the impor-tance of the proline-rich helix in the activation of SF (paper II, [100]).

Surprisingly we found that the L11NTD on its own could activate SF (see

Figure 6, paper II) and that this activation is ribosome-dependent and dependent on an unacylated tRNA in the A-site. Its ability to activate SF is not thought to be coupled to binding to the ribosome (paper II). Instead I think that L11NTD could be responsible for regulating SF activity by interactions with the SF before it is bound to the ribosome (but we were not able to confirm this). Another possible role for the L11NTD is to mediate interactions between ribosome-bound SF and the unacylated tRNA in the ribosomal A-site or interact with SF and the unacylated tRNA independently of each other.

SF and the sarcin/ricin loop in the ribosome In close contact with the important L11 and its binding site on the

ribosome is the SRL. Since SF was found to interact with the L11BD (Figure 10, blue) as previously discussed it was interesting to find footprints of SF also in the SRL (paper III and Figure 10, mangenta). The SRL has been shown to be involved in the binding of elongation factors [110] and their activation [133, 134] and the footprints found for SF in this region were found to partly overlaps with footprints of EF-G and EF-Tu [110, 135] (paper III and Figure 9, mangenta). SF was found to protect both the EF-G protected base A2660 and the EF-Tu protected base A2665 [110] in the SRL. When the backbone protected from hydroxyl radical cleavage by EF-G on the ribosome [136] was compared with the backbone protected by SF on the ribosome it was shown that both factors protect similar regions in L11BD, helix 89 and SRL. Ribosomes with EF-G or EF-Tu bound are inac-tive in synthesis of (p)ppGpp [137, 138] but there are contradictory results about whether SF and EF-G or EF-Tu can interact with the ribosome at the same time [6, 137, 138]. Richter et al suggested that EF-Tu, EF-G and SF recognizes non-identical sites on the ribosome [137] but in our footprinting results we can see that EF-G and EF-Tu have overlapping footprints with SF and it is therefore more likely that SF is unable to bind to ribosomes with bound EF-G like Wagner et al suggested [138]. To answer the question if this competition of binding sites is important for the regulation of protein synthesis during nutrient starvation and the stringent response more

36

observations must be done concerning the competition between SF and EF-G or EF-Tu.

SF and the A-site on the ribosome

The A-site and the activation of SF As already mentioned the A-site has been implicated in the function of SF

and it is known that an unacylated tRNA in the A-site is necessary for the ribosome-dependent activation of SF in E. coli (paper I, [4-6]). There are at least two possible options for how the unacylated tRNA enters the ribosomal A-site: it could enter the A-site by simple diffusion or as suggested by Richter et al, by interaction of SF with unacylated tRNA which increases the affinity of the tRNA for the ribosomal A-site [139].

The interaction between the unacylated tRNA and SF was suggested to be

dependent on the SFCTD since the SFNTD failed to be stimulated by un-acylated tRNA [22, 140]. There are however no proof of an interaction between the unacylated tRNA and the SF. In our study in paper I we showed that less tRNA is needed to reach maximal activation of SF when tRNA is added together with SF to the programmed ribosomes. I hypothesize that this could be due to that the interaction between SFCTD and tRNA can form more easily if tRNA is not already bound to the ribosome. As previously mentioned it is also possible that the L11NTD could be responsible for mediating the interaction between ribosome-bound SF and the unacylated tRNA in the ribosomal A-site (paper II).

The activity of SF has also been shown to be inhibited by the antibiotics,

viomycin and tetracycline which are known to interfere with A-site related functions [96, 111, 141-143] and this further strengthens the suggestion that the A-site is important for the activation of SF (paper I, [5, 144]). However a small amount of (p)ppGpp was found to be synthesized by SF even in the absence of unacylated tRNA, probably due to stabilization of SF by the ribosomal complex but when unacylated tRNA was added this resulted in a highly stimulatory effect of SF in agreement with previous studies [6]. We show in paper I that this effect was due to an unacylated tRNA bound in the A/A-state on the ribosome (Figure 6 in paper I).

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SF binds to domains involved in binding of tRNA In the light of the suggestion that SF can interact with tRNA and that the

function of SF is closely connected to the unacylated tRNA and the ribosomal A-site it was exciting to find that the binding of SF to the ribo-some resulted in the protection of the phosphate backbone in the A-loop (Figure 10, red) (paper III). Thus supporting the suggested interaction between SF and the unacylated tRNA in A-site of the ribosome on its own or possibly through the L11NTD (paper II). Other parts of the ribosome impli-cated in the binding of A-site tRNA [75] were also footprinted by SF bound to the ribosome. For instance footprints by SF were found in helix 89, that has been shown to run nearly parallel to the acceptor-arm and elbow of A-site tRNA making a minor groove interaction with the T stem at the top [75, 145].

The base A2602 (and C2601) in domain VI that were protected by SF

from chemical modification are suggested to be important for the interaction with the A-site tRNA [146, 147] and are suggested to be involved in the weak interaction with the 3´end of deacylated tRNA in the P-site [148] and also in peptide release [108, 109].

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Conclusion

The binding of SF is dependent on both ribosomes and a template for activation of SF. The unacylated tRNA is essential for the activation of SF and is suggested to bind in the A/A state on the ribosome.

L11 is important for regulating the function of SF although it is not

essential for the cell. Looking closer at the structure of ribosome-bound L11, the proline-rich helix located on the surface of the L11NTD could be a possible interaction site with SF since prolines are often found in close contact at protein-protein interactions sites. The L11NTD on its own was found to activate SF in a ribosome-dependent manner and I suggest that the L11NTD mediates an interaction between ribosome-bound SF and the un-acylated tRNA in the ribosomal A-site.

SF interacts with the 50S subunit of the ribosome and was found to

footprint nucleotides in domains where elongation factor etc. are found to bind, in close contact with the flexible L7/L12 stalk. Ribosome-bound SF also produced footprints within the L11BD, thus supporting the notion that SF is bound in close contact with L11.

The importance of the unacylated tRNA in the A-site was strengthened by the footprinting results which showed that SF protected nucleotides in domains in the 50S subunit that are involved in binding and interaction with the A-site bound tRNA, for instance the A-loop and helix 89. I suggest that the interaction between the unacylated tRNA and SF is dependent on the SFCTD.

SF also gave footprinted nucleotides in the highly conserved SRL shown

to be important for elongation factor binding and regulation of their activity. I suggest a model for the activation of SF where SF already is prebound

to some ribosomes in the cell. Upon binding of the unacylated tRNA to the A-site the L11NTD mediates an interaction between SF and the unacylated tRNA which leads to to a conformational change in SF that triggers the syn-thesis of (p)ppGpp.

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Future perspectives

• Further develop the method for purification of SF • Crystal structure of SF • Mutation studies of SF to determine which parts of the protein that

are essential for binding and activation of SF • Further analysis of the L11NTD and the proline-rich helix and its

role in activation of SF • Cryo-EM studies of ribosomal complexes containing SF and hope-

fully a crystal complex of a ribosomal complexes containing SF • Do human cells have a true stringent response? • Are the ribosomes in human cells affected by bacterial SF? • Investigate the possibility of using the acquired knowledge about

SF and stringent response to defeat resistant strains of bacteria for instance M. tuberculosis.

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Sammanfattning

Att kunna anpassa sig till sin omgivande miljö är en viktig egenskap hos en organism och saknar organismen denna förmåga riskerar den att dö ut. Bakterier har under evolutionen utvecklat en mängd olika mekanismer för att kunna överleva de olika stressfaktorer som de utsätts för under sin livscykel, till exempel brist på näring, syre, överskott av salt och påverkan av olika läkemedel. De flesta av dessa regleringsmekanismer sker på genetisk nivå och en av de mest studerade mekanismerna för överlevnad i bakterier är stringent respons. När en bakterie utsätts för näringsbrist så aktiveras stringent respons genom att ett protein som kallas för stringent faktor (SF) binder till en ribosom med en oacylerad tRNA (dvs utan aminosyra) bundet till A-site. Detta leder till aktivering av SF och produktion av ett alarmon, en signal molekyl som kallas för (p)ppGpp. ppGpp binder sedan till RNA polymeras som är ansvarigt för att översätta DNA till RNA och detta leder till en förändring av vilka gener som uttrycks. För en summering av förloppen under stringent respons, se figur 1 på sidan 14. Stringent respons kontrollerar ett flertal händelse-förlopp i en bakteries liv så som sporulering, bildandet av biofilm, förmågan att leva i symbios och även virulens, med andra ord så kan stringent respons påverka bakteriens fysiologi så att de kan överleva även under när de utsätts för olika stressfaktorer. Man har upptäckt att stringent respons är viktiga även för att patogena bakterier (dvs. sjukdomsalstrande bakterier) ska kunna överleva i värdorganismen. Om vi lär oss mer om stringent respons och på så sätt får mer kunskap om hur man kan förhindrar tillväxt av patogena organismer kan detta leda till nya behandlingsformer i framtiden, vilket är viktigt eftersom allt fler bakterier blir resistenta mot antibiotika.

Ribosomen är ett komplex som består av både RNA och proteiner och är ansvarig för proteinsyntesen i cellen dvs. produktionen av proteiner. Ribosomen består av två subenheter, den lilla subenheten (30S) och den stora subenheten (50S) samt en mängd ribosomala proteiner som tillsammans bildar 70S ribosomen.

I denna avhandling har jag studerat vad som krävs för att aktivera SF och

även var SF binder till ribosomen. Jag har renat fram ett koncentrerat rekombinant stringent faktor med hög renhet och genom att studera detta i

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ett in vitro-system (dvs. ett system utanför cellen) har jag kunnat visa att aktiveringen av SF är beroende av ribosomer och av ett tRNA bundet i A-site. Mutanter som saknar det ribosomala proteinet L11 (L11) kan inte aktivera SF och stringent respons. Jag har studerat hur L11 är involverat i aktiveringen av SF och fann att det är den flexibla N-terminala delen av L11 som är ansvarig för att aktivera SF troligen genom att binda till SF och tRNA antingen samtidigt eller oberoende av varandra. Genom att studera ribosomer med inbunden SF med en metod som kallas footprinting kunde man se att SF binder till 50S subenheten av ribosomen nära L11 vilket är intressant eftersom L11 är nödvändigt för aktivering av SF. Man kunde också se att SF binder till ribosomen i närheten av områden på ribosomen som är viktiga för bindning av faktorer under translationen och även för inbindning av tRNA till A-site.

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Acknowledgement

I would like to express my sincere gratitude to several people both inside and outside of the lab who, each in their own way, have supported and encourage me to complete this thesis. I would especially like to thank: Professor Odd Nygård, for introducing me to the research field and accept-ing me as a PhD-student. For all the fun discussions, not always scientific, during the years. My supervisor Lovisa Holmberg Schiavone, without your expertise, support and encouragement I would never have reached this point! Thanks for all the fun times! Birgit our labassistant, without you the lab is not complete! Thank you for all the nice hours in the lab, for all the technical help and for always being so positive and full of energy! Gunnar, for all the fun and sometimes crazy discussions about science and for always letting me get the last word ;)

Galina, thanks for sharing your knowledge about fabric, patterns and sawing. A new dress for the party perhaps?

Hossein, for all the discussions about life in general and for all the nice hours in the lab.

Ivo, for all the fun and crazy stories and �hyss�, you are the clown in our group! The former PhD students in the group, �the oldies� Sofia and Marika for introducing me to the group and for all the fun girl stuff outside the lab! All the people at floor 4 at Södertörns högskola, Maria, Kristina, Therese, Andrea, Kalle, Andreas, Anna, Fredrik, Sara, Inger, Lisbeth, Gaby and those already gone, thanks for creating a nice environment, for sharing their knowledge about science and for all the social lunches.

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Ida my collegue, ex kombo and dear friend, what would I have done without your support during these years! Thanks for always being there both for work and fun! Ulrika my colleague and friend, thanks for all the great lunches and all the pep talks. All my great friends from Lund University and especially; Estelle, thanks for all the encouragement, friendship, fun parties and for showing me that nothing is impossible. Caroline, so far away but still so close. Thanks for all the fun in the chemis-try lab, at the excursions, the hambo and for always being just a phone call away. Jenny, thanks for all the fun times and for good advice about life. Ellen, Therese, Hanna and Linda, thanks for all the pep talks, fun parties and nice dinners. Sara (& Mathias) my Linköping mates, thanks for the fun time during my time in Linköping and for staying in touch since then. Johan, my childhood friend, my dance partner and my private chef. Thanks for all the fun over the years and for always being there. No more lectures to go to! ;) Johan and Anders �Landskrona pågarna�, (Monica and Maria), thanks for all the fun times back home. Tias & Karin our �neighbours�, Anna & Zlatko, Sandra & Peter, Jenny & Tomas, Catharina & Fredric, Fredrik, Ella & Henrik, Anna & Mats and Cissi & Jeppe, thanks for all the fun times that have made me forget about science, even during the most stressful times.

Special thanks to my parents, Gertrud and Kurt for your endless love and encouragement, for all your support and for always believing in me. Without you none of this would have been possible.

Mattias, min kära lillebror, thanks for all the fun adventures during the years and for all your love and understanding, and Jessica Schön for all your sup-port.

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My parents in law, Camilla and Stellan thanks for accepting me into your family with open arms and for all your support during my years in Stock-holm.

Johanna & Donald, �Umeborna� thanks for all the fun trips and for all your support.

My godson Johan and my goddaughter Filippa for always putting a smile on my lips. And finally Niclas, �My first, my last, my everything and the answer to all my dreams You're my sun, my moon, my guiding star� Thanks for all your love and encouragement, for making me see the positive in thing even during stressful times and for all the adventures. I love you!

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References

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