Biophysical studies of the universally conserved NTPases HflX and YchF

Antibiotic resistance is becoming an increasing global health concern. The bacterial protein synthesis machinery, including the ribosome and its associated factors, are the target of over half of the clinically relevant antibiotics currently in use, highlighting the importance of cellular protein production as antibiotic target. The ribosome associated factors HflX and YchF are members of the GTPase superfamily. Their cellular functions are only poorly understood. The overarching goal of this thesis was to determine the location where HflX and YchF bind on the bacterial ribosome. Data presented here confirm that YchF interacts with the ribosomal A-site, while HflX is unique within the GTPase family being able to bind to both the ribosomal A-site and E-site. From this data and subsequent biochemical and biophysical studies, a mechanism for both HflX and YchF function during protein synthesis, specifically under stress conditions, is proposed

Novel inhibitors of the tRNA-dependent amidotransferase of "Helicobacter pylori" : Peptides generated by phage display and dipeptide-like compounds

Cette thèse présente la découverte de nouveaux inhibiteurs de l’amidotranférase ARNt-dépendante (AdT), et résume les connaissances récentes sur la biosynthèse du Gln-ARNtGln et de l’Asn-ARNtAsn par la voie indirecte chez la bactérie Helicobacter pylori. Dans le cytoplasme des eucaryotes, vingt acides aminés sont liés à leur ARNt correspondant par vingt aminoacyl-ARNt synthétases (aaRSs). Ces enzymes sont très spécifiques, et leur fonction est importante pour le décodage correct du code génétique. Cependant, la plupart des bactéries, dont H. pylori, sont dépourvues d’asparaginyl-ARNt synthétase et/ou de glutaminyl-ARNt synthétase. Pour former le Gln-ARNtGln, H. pylori utilise une GluRS noncanonique nommée GluRS2 qui glutamyle spécifiquement l’ARNtGln ; ensuite, une AdT trimérique, la GatCAB corrige le Glu-ARNtGln mésapparié en le transamidant pour former le Gln-ARNtGln, qui lira correctement les codons glutamine pendant la biosynthèse des protéines sur les ribosomes. La formation de l’Asn-ARNtAsn est similaire à celle du Gln-ARNtGln, et utilise la même GatCAB et une AspRS non-discriminatrice. Depuis des années 2000, la GatCAB est considérée comme une cible prometteuse pour le développement de nouveaux antibiotiques, puisqu’elle est absente du cytoplasme de l’être humain, et qu’elle est encodée dans le génome de plusieurs bactéries pathogènes. Dans le chapitre 3, nous présentons la découverte par la technique du « phage display » de peptides cycliques riches en tryptophane et en proline, et qui inhibent l’activité de la GatCAB de H. pylori. Les peptides P10 (CMPVWKPDC) et P9 (CSAHNWPNC) inhibent cette enzyme de façon compétitive par rapport au substrat Glu-ARNtGln. Leur constante d’inhibition (Ki) est 126 μM pour P10, et 392 μM pour P9. Des modèles moléculaires ont montré qu’ils lient le site actif de la réaction de transmidation catalysée par la GatCAB, grâce à la formation d’une interaction π-π entre le résidu Trp de ces peptides et le résidu Tyr81 de la sous-unité GatB, comme fait le A76 3’-terminal de l’ARNt. Dans une autre étude concernant des petits composés contenant un groupe sulfone, et qui mimiquent l’intermédiaire de la réaction de transamidation, nous avons identifié des composés qui inhibent la GatCAB de H. pylori de façon compétitive par rapport au substrat Glu-ARNtGln. Cinq fois plus petits que les peptides cycliques mentionnés plus haut, ces composés inhibent l’activité de la GatCAB avec des Ki de 139 μM pour le composé 7, et de 214 μM pour le composé 4. Ces inhibiteurs de GatCAB pourraient être utiles pour des études mécanistiques, et pourraient être des molécules de base pour le développement de nouvelles classes d’antibiotiques contre des infections causées par H. pylori.This thesis describes the discovery of inhibitors of a tRNA-dependent amidotransferase (AdT) and summarizes the present state of our knowledge about the two-step biosynthesis of Gln-tRNAGln and Asn-tRNAAsn in Helicobacter pylori. In eukaryotic cytoplasm, twenty amino acids (aa) are generally attached to their cognate tRNAs by twenty corresponding aminoacyl-tRNA synthetases (aaRSs). These enzymes have a high specificity, and their function is important to the proper decoding of mRNA. However, in a number of bacteria including H. pylori, GlnRS and/or AsnRS are absent. To synthesize Gln-tRNAGln, H. pylori first uses a noncanonical GluRS2 which is specific for tRNAGln to form Glu-tRNAGln; then the trimeric AdT (GatCAB) transforms Glu-tRNAGln into Gln-tRNAGln which is proper for protein biosynthesis. In a similar manner, the biosynthesis of Asn-tRNAAsn also takes place in H. pylori by using the same GatCAB and a canonical nondiscriminating AspRS. The widespread use of these indirect pathways among prominent human pathogens, and their absence in the mammalian cytoplasm, identify AdT as a promising target for the development of new and highly specific antimicrobial agents. By using phage display, we discovered several cyclic peptides rich in tryptophan and proline that inhibit H. pylori GatCAB. Peptides P10 (CMPVWKPDC) and P9 (CSAHNWPNC) are competitive inhibitors of GatCAB with respect to its substrate Glu-tRNAGln. The inhibition constants (Ki) of P10 and P9 are 126 and 392 μM, respectively. Their docking models revealed that they bind to the transamidation active site of GatB via π-π stacking interactions with Tyr81, as does the 3’-terminal A76 of tRNA. We also discovered two small dipeptide-like sulfone-containing inhibitors of H. pylori GatCAB by mimicking the intermediate of its transamidation reaction. Although they are much smaller than the cyclic peptides mentioned above, they are competitive inhibitors of GatCAB with respect to GlutRNAGln, with Ki values of 139 μM for compound 7 and 214 μM for compound 4. These inhibitors could be useful not only to study the reaction mechanisms of GatCAB, but also could be lead compounds for the development of a new class of antibiotics to treat infections caused by H. pylori

Prebiotic chemistry and the origin of life

The Sutherland group recently demonstrated the prebiotic synthesis of activated pyrimidine ribonucleotides as their 2',3'-cyclic phosphates, and these species are candidates for oligomerisation to RNA. These species hydrolyse to the corresponding 2'- and 3'-monophosphates and there is a need to discover prebiotically plausible ways to re-activate to the cyclic material. Previous methods have suffered from poor yields and/or derivatization of the nucleobase. This study describes a new multicomponent reaction that achieves highly efficient nucleotide activation and at the same time produces amino acid derivatives, also of importance in the origin of life. This reactivity is then further developed and utilised in the prebiotic synthesis of derivatives of glyceric acid 2- and 3-phosphate, used in the glycolysis pathway in contemporary biochemistry. Aminoacyl-RNA trimers are central to the RNA:coded peptides theory by Sutherland, whereby RNA replication and coded peptide synthesis are proposed to have emerged together in the origin of life. The aminoacylation of an RNA trimer is therefore investigated, again using a multicomponent reaction.With the prebiotic synthesis and re-activation of nucleoside-2',3'-cyclic phosphates shown, the oligomerisation of these species is now a major goal. The dry-state oligomerisation of these species using ethanolamine as catalyst is discussed. Key ethanolamine-adduct intermediates are identified, and the preference for the formation of natural [3'-5'] linkages produced by this type of oligomerisation is rationalised.The compartmentalisation of a primitive replicating genetic system is considered an important stage in the origin of life in order to overcome the high dilution of the oceans. Previous studies have focussed on long chain carboxylic acids for this purpose but these are unstable to the conditions required for RNA folding and catalysis, and only form bilayer vesicles at a specific pH. The final chapter investigates the prebiotic synthesis of a simple phospholipid amphiphile that has the potential to form more suitable lipid vesicles.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Potential prebiotic roles of (amino-)acylation in the synthesis and function of RNA

The Sutherland group recently demonstrated that from a mixture of oligoribonucleotide-2'- or 3'-phosphates the latter is chemoselectively acetylated. This is shown to mediate a template-directed ligation to give predominantly 3',5'-linked RNA that is acetylated at the ligation junction (acetyl-RNA). It was suggested that RNA emerged prebiotically via acetyl-RNA and also is proposed to have favourable genotypic properties due to greater propensity to form duplex structure. To study the properties of acetyl-RNA, their synthesis by solid-phase chemistry was required and described is the design of a 2'/3'-O-acetyl orthogonal protecting group strategy. Key to the orthogonal protecting group strategy is the use of (2-cyanoethoxy)carbonyl for the protection of the nucleobase exocyclic amines and a photolabile solid-phase linker group that allowed partial on-column deprotection. The synthesis of the 2'/3'-O-acetyl and 2'/3'-O-TBDMS phosphoramidites, in addition to preparation of a photolabile solid-phase support, are described. With the materials to hand the procedures for an automated synthesis of acetyl-RNA were optimised and several acetyl-RNA oligonucleotides were synthesised. The duplex stability of acetyl-RNA with up to four sites of 2'-O-acetylation were assessed by UV melting curve analysis. Remarkably, the acetyl groups caused a consistent decrease in Tm of between 3.0-3.2 °C. Thermodynamic parameters indicated a decrease in duplex stability that was consistent with a decrease in hydration of the minor groove resulting in a reduction of the stabilising hydrogen bonding network. The stability of a tetraloop was also found to decrease on acetylation. The acetylated- tetraloop it is able to form duplex at lower concentrations than the natural tetraloop. Additionally, it is more stable at high concentrations, indicating that acetyl-RNA favours duplex over other secondary structure. These properties are considered to give acetyl-RNA competitive advantage for their non-enzymatic replication. Aminoacylation of RNA is an important process in modern biology but the intermediacy of aminoacyl-adenylates is considered to be prebiotically implausible. A potentially prebiotic aminoacylation of nucleoside-3'-phosphates, selective for the 2'-hydroxyl, is presented. However, it was thought the aminoacylation yields could be improved and so a search for an alternative activator was conducted. Oligoribonucleotide-3'-phosphates were exposed to the aminoacylation conditions and selective aminoacylation at only the 2'-hydroxyl of the 3'-end was observed. In particular, the aminoacylation of a trimer lends support to Sutherland’s theory of a linked origin of RNA and coded peptide synthesis

Structural basis of translational recycling and bacterial ribosome rescue

In the last step of gene expression, a messenger RNA (mRNA) sequence is translated into a polypeptide. This highly regulated and dynamic process is carried out by the ribosome, a ribonucleoprotein complex composed of two unequal subunits. The translation cycle is initiated when the small ribosomal subunit (SSU) binds to an mRNA and recognizes the start codon of the open reading frame (ORF). Then the large ribosomal subunit (LSU) joins and the ribosome starts moving along the mRNA. A protein is synthesized until the ribosome reaches a stop codon. A cell needs thousands (prokaryotes) or millions (eukaryotes) of ribosomes for protein production and spends enormous amounts of energy on the assembly of this macromolecular machinery. Therefore, it is crucial to recycle the machinery after each successful round of translation. The recycling step allows release of mRNA, transfer RNA (tRNA) and the synthesized polypeptide from ribosomal subunits and subsequent binding of the next mRNA for protein synthesis. The first part of this dissertation includes studies of the highly conserved and essential ribosome recycling factor ATP binding cassette (ABC) Subfamily E Member 1 (ABCE1). In eukaryotes and archaea, ABCE1 binds the ribosome and in concert with an A-site factor and splits the ribosome into large and small subunits. ABCE1 harbors two nucleotide binding sites (NBSs), which are formed at the interface of two nucleotide binding domains (NBDs). Prior to this work, the ABCE1-bound pre-splitting complex, as well as the ABCE1-bound post-splitting complex, had been visualized by cryo-electron microscopy (cryo-EM) at medium resolution. This structural analysis combined with functional studies led to a model for the mechanism of the splitting event. ATP-binding and the closure of the NBSs lead to repositioning of the iron-sulfur cluster domain, which results in collision with the A-site factor and ribosome splitting. Yet, how conformational changes during the splitting event are triggered and communicated to the NBSs of ABCE1, was not understood. To gain molecular insights into this process, a structure of a fully nucleotide-occluded (closed) state of ABCE1 bound to the archaeal 30S post-splitting complex was solved by cryo-EM. At a resolution of 2.8 Å a detailed molecular analysis of ABCE1 was performed and confirmed by a combination of mutational and functional studies. This allowed to propose a refined model of how the ATPase cycle is linked to ribosome splitting and which role the different domains of ABCE1 play. In eukaryotes, the recycling phase is directly linked to translation initiation via the SSU. After being released from the mRNA 3’ end, the SSU can engage with another or even the same mRNA at the 5’ end. The recycling factor ABCE1 was found to be associated with initiation complexes, but whether it plays a role in initiation was not clear. Using cryo-EM, structures of native ABCE1-containing initiation complexes were solved and intensive 3D classification allowed to distinguish different stages of initiation, during which ABCE1 may play a role. Surprisingly, ABCE1 adopted a previously unknown state for ABC-type ATPases that was termed “hybrid state”. Here, the NBSI is in a half open state with ADP bound and the NBSII is in a closed state with ATP bound. Further, eukaryotic initiation factor 3j (eIF3j) was found to stabilize this hybrid conformation via its N-terminus. Since eIF3j had already been described to assist ABCE1 in ribosome dissociation, in vitro splitting assays were performed demonstrating that eiF3j indeed actively enhances the splitting reaction. On top of this, the high-resolution structure allowed to describe the interaction network of eIF3j with the ribosome, initiation factors (IFs), and ABCE1. Independent of ABCE1, the structures presented here allowed to provide an improved molecular model of the human 43S pre-initiation complex (PIC) and to analyze its sophisticated interaction network. In particular, new molecular insights into the large eIF3 complex encircling the 43S PIC, and the eIF2 ternary complex delivering the initiator tRNA are provided. Equally important as canonical recycling is the recognition and recycling of ribosomes that result from translational failure. Aberrant translation elongation and ribosome stalling can be caused by a plethora of different stresses. In bacterial cells, multiple rescue systems are known such as trans-translation or alternative ribosome rescue factor-mediated termination, which act on ribosome nascent chain complexes with an empty A-site (non-stop complexes). It has been a long standing question how ribosomes that are stalled in the middle of an ORF (no-go complexes) are recognized and recycled. The second part of this dissertation reports a new bacterial rescue system that acts on no-go complexes. In eukaryotes, the concept of ribosome collisions as a trigger for ribosome rescue has been studied extensively. Here, it was found that a similar mechanism exists in bacteria and thus a structural analysis of collided disomes in E. coli and B. subtilis was conducted. In a genetic screen, the endonuclease SmrB was identified as one candidate for a collision sensor. Structural analysis of SmrB-bound disomes elucidated how this rescue factor is recruited to collided ribosomes. Its SMR domain binds to the disome interface between the stalled and the collided ribosome in close proximity to the mRNA and in a position ideal to perform endonucleolytic cleavage. Such cleavage then results in non-stop complexes that can be recycled by the pathways mentioned above. In conclusion, this work provides mechanistic insights into how a cell distinguishes stalled ribosomes from actively translating ribosomes and characterizes a novel ribosome rescue pathway

Importance of single molecular determinant in bacterial tryptophanyl-tRNA synthetase fidelity in expanded genetic code

Nonnatural amino acid incorporation is a valuable method for introducing novel chemical functional groups into proteins. For this method, an orthogonal arninoacyl-tRNA synthetase (AARS) and a cognate tRNA that suppress an encoded stop codon are introduced into the cell (these components are required to be orthogonal). Nonnatural amino acids (NAAs) are usually incorporated efficiently by using Methanocaldococcus jannaschii tyrosyl-AARS/tyrosyl-tRNA pair (Mj TyrRS/A// tRNATyr) in Escherichia coli. High translation fidelity of a synthetase is achieved by site-directed mutagenesis of the competent active site residues. The active site of the mutant Mj TyrRS displays two crucial mutations of residues that interact with the tyrosine hydroxyl group (-OH). We demonstrated that the fidelity of the synthetase would be affected if only one of these residues is restored and does not undergo mutagenesis. We found a similar situation in the case of tryptophanyl-AARS (TrpRS) from Bacillus subtilis. TrpRSs are structurally similar to TyrRSs, but there is one crucial residue of the substrate specificity. We uncovered that a NAA system developed to incorporate 5-hydroxytryptophan (5-OH Tip) in mammalian cells does not contain this crucial residue mutation in the TrpRS active site. Even though this mutant TrpRS was designated as a high fidelity enzyme, our results challenge this conclusion. Expanded genetic codes have a similar capacity to impact science as has standard mutagenesis. Only the full impact of the method will be achieved if the technology functions in all cell types. Therefore, our reinvestigation of the first report of expanded genetic code in mammalian system is critical to ensuring that the field is on the optimum path to realising the full potential of the method

Dynamics of Stop-codon Recognition by Release Factor 1

Translation of an mRNA template into its corresponding protein is necessarily a highly accurate process. In all organisms, this translation is performed by the universally conserved macromolecular machine, the ribosome. However, the mechanisms through which the ribosome is able to regulate translation, and therefore ensure its fidelity, are not well understood. Often these types of mechanisms, which ensure molecular fidelity, utilize multiple, transient states over which cognate and non-cognate substrates are discriminated multiple times. However, such transient and/or rarely populated states are difficult to study by conventional, ensemble experimental techniques. In this thesis, single-molecule fluorescence resonance energy transfer (smFRET), which alleviates many of these limitations, is used in order to interrogate the dynamics of a translation factor, release factor 1 (RF1), and how they are organized to ensure accurate and efficient recognition of stop-codons during the termination stage of translation. In order to observe the dynamics of the RF1 binding and codon discrimination processes with smFRET, a relatively high concentration of fluorophore-labeled RF1 must be used in order to observe significant binding to sense-codons; however, such high concentrations are not accessible with traditional smFRET total internal fluorescence microscopy. Therefore, in Chapter 2 a novel approach to breaking this concentration barrier is presented, in which robustly-passivated gold-based nanoaperture arrays are developed to limit the excitation volume used in smFRET measurements of RF1. Unfortunately, as in the case of RF1 binding to sense-codon programmed ribosomes, many of the ribosomal dynamics that are in principal observable using smFRET are too fast to observe using current wide-field detectors. Therefore, Chapter 3 investigates the precision and accuracy with which transient conformational dynamics can be quantified using single-molecule techniques such as smFRET. As a case study, these approaches were used to analyze the dynamics of the GS1-GS2 equilibrium of the pretranslocation (PRE) ribosome--a situation where transient intermediate states that can be observed using single-particle cryo-electron microscopy are not seen using smFRET. In Chapter 4, a novel computational method is developed to address such temporally-limited single-molecule data, and in doing so, it is used to analyze the structural contributions of tRNA to ribosomal transition state energy barriers using temperature-dependent smFRET with temporal super-resolution. The temperature-dependence of reaction rate constants is governed by the underlying thermodynamic landscape of the molecular system. To investigate the energy landscape over which the PRE ribosome operates, temperature-dependent smFRET experiments were performed on PRE complexes containing different tRNAs. By investigating the relative temperature-dependence of the rate constants involved in the GS1 - GS2 equilibrium as a function of tRNA identity, nascent polypeptide chain presence, and A and P site occupation, relative thermodynamic contributions of the different structural elements were quantified. Unfortunately, this investigation was complicated by fast rate constants which approach the time resolution limitations of smFRET TIRF experiments, especially with the increased temperatures used in these experiments. Additionally, it is complicated by the heterogeneity within the ensemble of ribosomes that is created when some of the enzymatically-prepared ribosomal complexes fail to undergo, or undergo additional rounds of translation. To overcome these complications, a novel computational method to achieve temporal super-resolution. This method uses Bayesian inference for the analysis of sub-temporal resolution data (BIASD). By integrating this approach with a Bayesian variational mixture model, the fast dynamics of heterogenous populations can be accurately and precisely quantified. This then allowed the contributions of the structural differences that the various tRNA make to the underlying PRE complex energy landscape to be determined. The conformational dynamics that regulate the binding affinity and codon discrimination ability of RF1 are investigated in Chapter 5. During the elongation stage of translation, class I release factors compete with aminoacyl-tRNAs to interrogate the mRNA triplet-nucleotide codon that is located in the ribosomal aminoacyl-tRNA binding (A) site. To avoid deleterious effects, class I RFs must be able to accurately discriminate stop-codons from sense-codons, only triggering the termination stage of translation and catalyzing the release of the nascent polypeptide chain from the peptidyl-tRNA located in the ribosomal peptidyl-tRNA binding (P) site upon recognition of a stop-codon. Despite its importance for ensuring the accuracy of gene expression, the high fidelity mechanism through which class I RFs discriminate sense codons remains elusive. Using smFRET, the kinetics with which a fluorophore-labeled, bacterial RF1 binds to the A site of bacterial ribosomal release complexes carrying a fluorophore-labeled peptidyl-tRNA in the P site and either a stop-codon, or a sense-codon that differs from a stop-codon by a single nucleotide (i.e., a near-stop codon) programmed in the A-site are investigated. The results of these experiments, as well as analogous experiments performed using RF1 mutants or antibiotic inhibitors of RF1 function, reveal that RF1 binding affinity and codon discrimination occurs via a multistep process. Taken together with molecular dynamics simulations of wildtype and mutant RF1, these data demonstrate how the conformation dynamics of the switch loop modulate RF1 binding affinity and codon discrimination--enabling the elucidation of some of the molecular details through which class I RFs ensure the integrity of translation elongation and the fidelity of translation termination