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In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fmet-tRNA fMet . Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entry channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 A resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance. Structural analysis of the A2058-dimethylated and unmethylated 70S ribosome complex alone and in combination with macrolides reveals the role of the desosamine moiety of macrolides in drug binding and resistance.

Proline‐rich antimicrobial peptides expressed in insects are primarily active against Enterobacteriaceae. Mechanistically, they target the bacterial (70S) ribosome after partially transporter‐based cellular uptake, as revealed for Api137 and Onc112 on Escherichia coli. Following molecular modeling indicating that the Onc112 contact site is conserved among the ribosomes of high‐priority pathogens, the ribosome binding of Api137 and Onc112 was studied. The dissociation constants (K d) of Onc112 were ∼75 nmol/L for Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, 36 nmol/L for Pseudomonas aeruginosa, and 102 nmol/L for Staphylococcus aureus, thus indicating a very promising lead structure for developing broad‐spectrum antibiotics. Api137 bound weaker with K d values ranging from 155 nmol/L to 13 μmol/L. For most bacteria, the antibacterial activities were lower than predicted from the K d values, which was only partially explained by their ability to enter bacterial cells. Other factors limiting the activity expected from the ribosome binding might be off‐target binding., Resisting resistance: Proline‐rich antimicrobial peptides Api137 and especially Onc112 bind strongly to the bacterial (70S) ribosome, even to ribosomes of pathogens that appear to be resistant in standards antibiotic tests. The presented data indicate that Onc112 in particular might represent a valid lead structure to develop broad‐spectrum antibiotics relying on a novel bactericidal mechanism.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

Significance Sarecycline is the first narrow-spectrum tetracycline-class antibiotic that was recently approved by the FDA for the clinical treatment of acne vulgaris. In this work, we determined two (2.8-Å and 3.0-Å) X-ray crystal structures of sarecycline bound to the initiation complex of the bacterial 70S ribosome and found that this antibiotic inhibits bacterial ribosome in part using a mechanism of direct mRNA contact, which has not been reported for any other tetracyclines so far. Moreover, our structural analysis rationalizes why sarecycline is able to overcome one of the most common mechanisms of resistance to tetracyclines among pathogenic bacteria. Thus, this work provides mechanistic insights into the function of the tetracycline class of antibiotics on the ribosome with direct clinical relevance., Sarecycline is a new narrow-spectrum tetracycline-class antibiotic approved for the treatment of acne vulgaris. Tetracyclines share a common four-ring naphthacene core and inhibit protein synthesis by interacting with the 70S bacterial ribosome. Sarecycline is distinguished chemically from other tetracyclines because it has a 7-[[methoxy(methyl)amino]methyl] group attached at the C7 position of ring D. To investigate the functional role of this C7 moiety, we determined the X-ray crystal structure of sarecycline bound to the Thermus thermophilus 70S ribosome. Our 2.8-Å resolution structure revealed that sarecycline binds at the canonical tetracycline binding site located in the decoding center of the small ribosomal subunit. Importantly, unlike other tetracyclines, the unique C7 extension of sarecycline extends into the messenger RNA (mRNA) channel to form a direct interaction with the A-site codon to possibly interfere with mRNA movement through the channel and/or disrupt A-site codon–anticodon interaction. Based on our biochemical studies, sarecycline appears to be a more potent initiation inhibitor compared to other tetracyclines, possibly due to drug interactions with the mRNA, thereby blocking accommodation of the first aminoacyl transfer RNA (tRNA) into the A site. Overall, our structural and biochemical findings rationalize the role of the unique C7 moiety of sarecycline in antibiotic action.

Acinetobacter baumannii is a severe nosocomial threat largely due to its intrinsic antibiotic resistance and remarkable ability to acquire new resistance determinants. The bacterial ribosome serves as a major target for modern antibiotics and the design of new therapeutics. Here, we present cryo-EM structures of the A. baumannii 70S ribosome, revealing several unique species-specific structural features that may facilitate future drug development to combat this recalcitrant bacterial pathogen., Antimicrobial resistance is a major health threat as it limits treatment options for infection. At the forefront of this serious issue is Acinetobacter baumannii, a Gram-negative opportunistic pathogen that exhibits the remarkable ability to resist antibiotics through multiple mechanisms. As bacterial ribosomes represent a target for multiple distinct classes of existing antimicrobial agents, we here use single-particle cryo-electron microscopy (cryo-EM) to elucidate five different structural states of the A. baumannii ribosome, including the 70S, 50S, and 30S forms. We also determined interparticle motions of the 70S ribosome in different tRNA bound states using three-dimensional (3D) variability analysis. Together, our structural data further our understanding of the ribosome from A. baumannii and other Gram-negative pathogens and will enable structure-based drug discovery to combat antibiotic-resistant bacterial infections.

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001, eLife digest Proteins fold into structures that are determined by the order of the amino acids that they are built from. These structures enable the protein to carry out its role, which often involves interacting with other proteins. Chemical cross-linking coupled with mass spectrometry (CXMS) is a powerful method used to study protein structure and how proteins interact, with a benefit of stabilizing and capturing brief interactions. CXMS uses a chemical compound called a linker that has two arms, each of which can bind specific amino acids in a protein or in multiple proteins. Only when the regions are close to each other can they be “cross-linked” in this way. After cross-linking, the proteins are cut into small pieces known as peptides. The cross-linked peptides are then separated from the non cross-linked ones and characterized. Although CXMS is a popular method, there are aspects about it that limit its use. It does not work well on complex samples that contain lots of different proteins, as it is difficult to separate the cross-linked peptides from the overwhelming amounts of non cross-linked peptides. Also, although it can be used to detect changes in the shape of a protein, which are often crucial to the protein's role, the method has not been smoothed out. Tan, Li et al. have now developed a new cross-linker called Leiker that addresses these limitations. Leiker cross-links the amino acid lysine to another lysine, and contains a molecular tag that allows cross-linked peptides to be efficiently purified away from non cross-linked peptides. As part of a streamlined workflow to detect changes in the shape of a protein, Leiker also contains a region that can be labeled. Analysing a bacterial ribosome, which contains more than 50 proteins, showed that Leiker-based CXMS could detect many more protein interactions than previous studies had. These included interactions that changed too rapidly to be studied by other structural methods. Tan, Li et al. then applied Leiker-based CXMS to the entire contents of bacterial cells at different stages of growth, and identified a protein interaction that is only found in growing cells. In future, Leiker will be useful for analyzing the structure of large protein complexes, probing changes in protein structure, and mapping the interactions between proteins in complex mixtures. DOI: http://dx.doi.org/10.7554/eLife.12509.002

Protein synthesis on the ribosome involves a number of external protein factors that bind at its functional sites. One key factor is the elongation factor G (EF-G) that facilitates the translocation of transfer RNAs between their binding sites, as well as advancement of the messenger RNA by one codon. The details of the EF-G/ribosome diffusional encounter and EF-G association pathway still remain unanswered. Here, we applied Brownian dynamics methodology to study bimolecular association in the bacterial EF-G/70S ribosome system. We estimated the EF-G association rate constants at 150 and 300 mM monovalent ionic strengths and obtained reasonable agreement with kinetic experiments. We have also elucidated the details of EF-G/ribosome association paths and found that positioning of the L11 protein of the large ribosomal subunit is likely crucial for EF-G entry to its binding site. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 616–627, 2011.

The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 A resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

Wenn sich Bakterien der stationären Phase nähern, reduziert sich ihre Teilungsrate, die metabolischen Aktivitäten werden zurück gefahren und die Translationsaktivität der Ribosomen nimmt rapide ab. Letzteres bedingt den wohlbekannten Effekt, daß Ribosomen, isoliert von Zellen aus der stationären Phase, nur eine geringe Aktivität in vitro haben. Wir haben nur geringe Kenntnis von den Ursachen der reduzierten ribosomalen Aktivität („silencing“) während der stationären Phase und unter Stressbedingungen. Faktoren wie RMF, HPF und sein Homolog PY wurden vorgeschlagen, über Dimerisation von 70S inaktive 100S Partikel zu bilden. Jedoch gibt es keine Übereinstimmung über die Rolle und das Auftauchen dieser Partikel. Ferner ist bekannt, daß die Entfernung des RMF Gens die Lebensfähigkeit von E. coli Zellen in der stationären Phase verschlechtert. Weiterhin wurden 100S Partikel auch in logarithmischer Phase beobachtet, was heißen mag, daß die 100S Partikel eine andere Rolle spielt oder weitere Funktionen besitzt. Wir präsentieren hier eine Studie des kürzlich von uns und mit meiner Beteiligung beschriebenen „Ribosome Silencing Factor“ (RsfS, früherer Name YbeB), ein Protein das mit Ribosomen assoziiert ist. RsfS kommt fast in allen Bakterien, Mitochondrien und Chloroplasten vor und bindet an L14 der großen, ribosomalen Untereinheit, eines der am besten konservierten Proteine des Ribosoms. Die Wechselwirkung von RsfS mit L14 ist vom Bakterien bis zum Menschen konserviert. Wir zeigen, daß RsfS wichtig für das Überleben ist, wenn immer die Wachstumsrate herunter gefahren werden muß, d.h. während des Übergangs von der logarithmischen zur stationären Phase oder vom reichen zum armen Medium. Im letzteren Fall ist das Wachstum blockiert, bis es nach etwa 15 h langsam wieder Fahrt aufnimmt. Entfernung des RsfS Gens erhöht die Translationsaktivität in der stationären aber nicht in der logarithmischen Phase. In vitro hemmt RsfS und sein mitochondriales Homolog die Translation über die Bindung an L14 in der großen ribosomalen Untereinheit und blockiert damit die 70S Bildung aus Untereinheiten oder dissoziiert leere 70S Ribosomen. Interessanterweise wird die Effizienz zur Dissoziation empfindlich gestört, wenn programmierte Ribosomen tRNAs tragen. RsfS hemmt nicht die Translation von chemisch vernetzten 70S Ribosomen, woraus wir schließen, daß die Translationshemmung zum wesentliche Teil auf Dissoziation der 70S Ribosomen bzw. Hemmung der Assoziation der ribosomalen Untereinheiten zurückgeführt werden kann. Wir haben auch RsfS mit den Faktoren RMF, PY und HPF genetisch und funktionell verglichen, um die jeweilige Bedeutung und eine mögliche Kooperation zu entdecken. Wir fanden, (i) daß 100S Bildung keine obligates Merkmal der stationären Phase ist, (ii) daß der schwere Phänotyp des ΔrsfS Stammes mit entsprechenden KO-Mutanten der drei anderen Faktoren nicht beobachtet wird, (iii) daß die Lebensfähigkeit von ΔrsfS Zellen und besonders der Δrmf Zellen, aber nicht der Δhpf or Δpy stark eingeschränkt ist, und daß schließlich in vitro RsfS die stärkste Translationshemmung sowohl bei natürlichen mRNAs als auch bei hoch definierten Elongationsexperimenten zeigt. Die Hemmung der anderen Faktoren ist additiv, nicht kooperativ. Zusammengefaßt zeigen unsere Daten, daß RsfS eine Schlüsselrolle für das ribosomale “silencing” hat, wobei es von den anderen Faktoren unterstützt wird., Bacterial cells approaching stationary growth phase reduce division rates, cut back metabolic activities and thus decrease protein translation causing the well-known effect that ribosomes isolated from stationary growth phase show a low translational activity. We have a scarce knowledge about the mechanism of ribosome silencing during stationary growth phase or under stress condition. HPF, its homolog PY and RMF have been proposed to bring translation to a halt by dimerization of 70S ribosomes into 100S particles. However, there is no consensus about the function or occurrence of 100S particles. Deletion of RMF decreases the viability of E. coli in stationary growth phase. On the other hand, 100S particles have been observed also in logarithmic growth phase, suggesting that 100S particles have a different role than ribosome silencing. Here we present a study of the recently (with my participation) described Ribosome Silencing Factor S (former name YbeB), a protein associated with ribosomes. RsfS is present in almost all bacteria, mitochondria and chloroplasts and binds to protein L14, one of the most conserved proteins of the large ribosomal subunit. This interaction is conserved from bacteria to man. We demonstrate that RsfS is important for cell survival, whenever the growth rate has to be decreased, i.e. during the transition from the logarithmic growth phase to the stationary growth phase and from rich to poor media. In the latter case ΔrsfS strain stops growing for about 15 h before growth is launched again. Deletion of RsfS gene increases the translation activity during stationary growth phase but not during logarithmic growth phase. In vitro RsfS and its mitochondrial homolog inhibits translation by binding to 50S protein L14 and thus i) inhibits 70S formation from subunits and ii) dissociates empty 70S ribosomes. Interestingly, the efficiency of 70S dissociation decreases, when 70S is programmed with tRNA at the P site. RsfS does not inhibit translation of non dissociable 70S suggesting that ribosome silencing by RsfS is mediated predominantly via 70S dissociation or anti- association of the ribosomal subunits. We also compared RsfS with RMF, PY and HPF to elucidate the importance and possible interplay between these factors in ribosome silencing. We found that i) 100S formation is not an obligatory feature of stationary-phase E. coli cells; ii) the strong phenotype of ΔrsfS strain, viz. a block of growth for about 15 h after transfer from rich to poor media, is not seen with the knock-out of one of the other factors; (iii) viability at stationary phase is impaired in ΔrsfS cells and even stronger in Δrmf cells, but not in Δhpf or Δpy cells; (iv) RsfS is the only factor that impairs translation in stationary growth phase; (v) in vitro RsfS shows the strongest inhibition in both translation of natural mRNAs and in highly resolved elongation assays; the inhibition of the other factors is additive, not cooperative. Collectively, the data indicate that RsfS plays a key role in silencing the ribosomal activity under conditions characterized by a reduced growth rate, and that it is supported by the other silencing factors.

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation., Author Summary Translation is the process by which a ribosome converts the sequence of a messenger RNA (mRNA)—produced from a gene—into the sequence of amino acids that comprise a protein. Bacterial ribosomes each have one large and one small subunit: the 50S and 30S subunits. Initiation of translation entails selection of an mRNA, identification of the correct starting point from which to read its code, and engagement of the initial amino acid carrier (tRNA). These events take place in the 30S subunit and require the presence of three initiation factors (IF1, IF2, IF3). Formation of this 30S initiation complex precedes joining with the 50S subunit to assemble the functional ribosome. By using a cryo-electron microscopy approach to visualize the structures without fixation or staining, we have determined the structure of a complete 30S initiation complex and identified the positions and orientations of the tRNA and all three initiation factors. We found that the presence of the initiation factors and tRNA induces rotation of the head relative to the body of the 30S subunit, which may be essential for rapid binding to the 50S subunit and for regulating selection of the mRNA. IF3 had not been seen previously in the context of the 30S structure and its visualization gives insight into a potential role in preventing association of the two ribosomal subunits. These findings are important for understanding how the interplay of elements during the early stages of translation selects the mRNA and regulates formation of functional ribosomes.

The canonical initiation mode starts with the small ribosomal subunit, which with help of initiation factors and the initiator tRNA finds the start signal for protein synthesis on an mRNA. However, there are many observations in the literature that cannot easily be reconciled with this initiation mode. We found that a resolution of these difficulties can be provided with an alternative initiation mode, the so-called 70S-scanning mode. In this thesis we provide in vivo evidences supporting it. Towards this end we identify features that critically participate in this process. 1\. According to general wisdom the initiation factor IF1 binds to 30S subunits helping IF2 and IF3 to select the ribosomal binding site on the mRNA. The factor is believed to leave the 30S subunit upon association with the large ribosomal subunit. Therefore, it should never be present on 70S ribosomes. We determined the ribosome location of IF1 using cytosol-profiles on sucrose gradients and identified IF1 via Western blotting. It was strikingly found that IF1 is present specifically on 70S and polysomes rather than on 30S subunits. IF3 is thought to be an anti-association factor and thus should not be able to bind to 70S ribosomes. We found it on 30S subunits as expected, but 1/3 of the amount was present on 70S and significant amounts even on disomes. Obviously, the functional horizon of these initiation factors is wider than thought before. 2\. IF1 is an essential factor. To study its function in vivo we constructed a strain, where the chromosomal IF1 was knocked out and the IF1 gene present on a plasmid after an inducible AraB promoter (in the presence of arabinose IF1 is expressed, in the presence of glucose it is not). The effects of insufficient IF1 amounts can be summarized as follows: (i) A slowed growth rate with doubled generation time. (ii) Serious defects in 50S assembly leading to accumulation of 50S-precursors, accumulation of 30S subunit and strikingly poor polysome pattern. Obviously, the block in 50S formation reduces the formation of 70S and thus explains the indispensability of IF1 for bacterial cell survival. The specific defects of the 50S assembly are consistent with an assumption of involvement of IF1 in scanning ribosomes leading to stoichiometric synthesis of ribosomal proteins. 3\. Our assumption was that IF1 is essential for the 70S scanning initiation. To test this we constructed a bicistronic mRNA expressing Renilla and Firefly luciferase, respectively. The hypothesis predicts that the first cistron might be preferentially initiated in the canonical mode, whereas for the second expression the IF1-dependent scanning mechanism would be more important. Precisely this was observed: The expression of the second cistron was 4.5 times reduced, when the cells were starved for IF1. Remarkably, the expression of the first cistron was practically not affected at all by reducing IF1 amounts. Western blotting showed that this expression-reduction was accompanied by reducing the IF1 amounts for about 50%. This observation distinctly presents the evidence of involvement of IF1 in sliding 70S ribosomes, leading to translation initiation of second cistron. 4\. Eventually we demonstrated that the 70S-scanning type of initiation also exists for the expression of monocistronic mRNAs. With a strong secondary structure in the 5’-UTR abolishing the scanning mode we observed that the expression of the reference protein GFP was not affected by various levels of IF1, whereas without secondary structure allowing both initiation modes the expression was strongly IF1 dependent. In summary we provided strong evidence that IF1 is a 70S specific factor and participates crucially in the 70S-scanning type of initiation. Since the 70S-scanning mode is importantly involved in the expression of both mono- and polycistronic mRNAs it might be even the prevailing initiation mode in the bacterial cell., Das Standardmodell der Initiation beginnt mit der kleinen Untereinheit, die mit Hilfe der Initiationsfaktoren und der Initiator fMet-tRNA das Startsignal für die Proteinsynthese auf einer mRNA findet. Jedoch sind einige Beobachtungen nicht einfach mit dem Standardmodell in Einklang zu bringen. Wir prüfen in der vorliegenden Arbeit eine Hypothese, nach der neben dem Standardmodell eine zweite Initiationsart existiert, das sogenannte 70S- Scanning Modell, das die Ungereimtheiten befriedengend erklären kann. Die erzielten Ergebnisse der in vivo Untersuchungen unterstützen das Modell: 1\. Nach allgemeiner Ansicht bindet der Initiationsfaktor IF1 an die kleine Untereinheit und unterstützt die Faktoren IF2 und IF3, die ribosomale Bindungsstelle für den Start der Proteinsynthese auf einer mRNA zu finden. Die Annahme ist, dass IF1 die 30S Untereinheit nach Assoziation der großen verläßt. Daraus folgt, daß dieser Faktor nicht auf 70S Ribosomen anzutreffen sein soll. Wir bestimmten die IF1 Lokalisation auf ribosomalen Partikeln mittels Cytosol-Profilen auf Sucrose-Gradienten und anschließender IF1 Identifikation mittels Western-Blot. Zu unserer Überraschung fanden wir IF1 ausschließlich auf 70S und zu einem geringen Anteil auf Disomen, aber nicht auf der kleinen Untereinheit. IF3 als Antiassoziationsfaktor wird für einen spezifischen 30S Faktor gehalten, der nicht auf 70S Ribosomen anzutreffen sein soll. Unsere Western-Analyse zeigte, dass wie erwartet ein großer Teil des IF3 wie erwartet an die 30S Untereinheit bindet, aber etwa ein Drittel der Menge an 70S Ribosomen bindet. Es folgt daraus, dass der funktionelle Horizont beider Faktoren offenbar weiter reicht als allgemein angenommen. 2\. IF1 ist ein essentieller Faktor. Um dessen Funktionen in vivo zu testen, haben wir einen E. coli Stamm konstruiert, dem das chromosomale IF1-Gen fehlt und der Zelle auf einem Plasmid angeboten wird, dessen AraB Promoter die IF1 Synthese an- und ausschalten kann (in Gegenwart von Arabinose wird IF1 synthetisiert, während Glucose die Synthese abschaltet). Die Effekte einer IF1 Verarmung können folgendermaßen zusammengefaßt werden: (i) Eine 50% Reduktion der IF1 Menge halbiert die Wachstumsrate. (ii) Die Bildung der großen 50S Untereinheit ist schwer geschädigt, 50S Vorstufen werden angehäuft, 30S Untereinheiten akkumulieren mit dem Ergebnis, dass 70S Ribosomen sowie Polysomen deutlich vermindert sind. Die spezifischen Defekte des 50S Aufbaus können damit erklärt werden, daß IF1 an einem 70S-Scanning Initiations-modus beteiligt ist, was zu einer stöchiometrischen Synthese der ribosomalen Proteine führt. 3\. Unsere Annahme, dass IF1 wichtig für eine 70S-Scanning Initiation ist, wurde folgendermaßen getestet: wir konstruierten eine bi-cistronische mRNA, mit der die Renilla und die Feuerfliegen Luziferase exprimiert werden kann. Die produzierte Menge beider Luziferasen können in einem Ansatz ohne Überlappung getestet werden. Unsere Hypothese sagt voraus, dass das erste Cistron vornehmlich nach dem kanonischen 30S Modell initiiert wird, während bei der Expression des zweiten Cistrons der IF1 abhängige Scanning-Modus deutlicher beteiligt ist. Genau das wurde beobachtet: Eine IF1 Reduktion um 50% reduzierte die Translation des zweiten Cistrons um das 4,5 fache, während interessanterweise die Translationsleistung am ersten Cistron durch reduzierten IF1 Gehalt gar nicht beeinträchtigt wurde. Dieser Befund ist eine deutliche Unterstützung des Scanningmodells und belegt zum ersten Mal, dass IF1 wahrscheinlich ein spezifischer 70S Initiationsfaktor ist und eine geringe Rolle – wenn überhaupt – bei der kanonischen 30S Initiation spielt. 4\. Schließlich haben wir gezeigt, dass der 70S-Scanning Typ auch bei der Translation von mono-cistronischen mRNAs beteiligt ist. Bei einer mRNA mit einer starken Sekundärstruktur an der 5’-UTR, die 70S-Scanning verhindert aber eine 30S abhängige Initiation erlaubt, ist die Expression von GFP unabhängig von der IF1 Menge in der Zelle, während ohne Sekundärstruktur die GFP Synthese stark von der IF1 Menge abhängig war. Zusammengefasst, haben wir sehr starke Hinweise, dass IF1 ein spezifischer 70S-Scanning Initiationsfaktor ist, der eine bedeutende Rolle bei diesem neuen Typ der bakteriellen Initiation spielt. Da die 70S-Scanning Initiation sowohl bei der Initiation von poly- als auch mono-cistronischen mRNAs eine Rolle spielt, könnte dieser Initiationstyp sogar der in der bakteriellen Zelle vorherrschende sein.

AcPhe2-tRNA(Phe) synthesized in 70S ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. A detailed study of the kinetics of the puromycin reaction, its comparison with that of spontaneous translocation, the use of antibiotic viomycin as an effective inhibitor of spontaneous translocation revealed that, besides spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction per se triggers conformational changes in the ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl-site of the peptidyltransferase center. This is detected functionally as the ability of such an A site bound peptidyl-tRNA to react with puromycin. This reaction is highly pronounced at elevated (25 degrees C) temperature but can be hardly detected at 0 degrees C.