Crucial contribution of the multiple copies of the initiator tRNA genes in the fidelity of tRNAfMet selection on the ribosomal P-site in Escherichia coli

The accuracy of the initiator tRNA (tRNAfMet) selection in the ribosomal P-site is central to the fidelity of protein synthesis. A highly conserved occurrence of three consecutive G–C base pairs in the anticodon stem of tRNAfMet contributes to its preferential selection in the P-site. In a genetic screen, using a plasmid borne copy of an inactive tRNAfMet mutant wherein the three G–C base pairs were changed, we isolated Escherichia coli strains that allow efficient initiation with the tRNAfMet mutant. Here, extensive characterization of two such strains revealed novel mutations in the metZWV promoter severely compromising tRNAfMet levels. Low cellular abundance of the chromosomally encoded tRNAfMet allows efficient initiation with the tRNAfMet mutant and an elongator tRNAGln, revealing that a high abundance of the cellular tRNAfMet is crucial for the fidelity of initiator tRNA selection on the ribosomal P-site in E. coli. We discuss possible implications of the changes in the cellular tRNAfMet abundance in proteome remodeling

Single-Molecule Analysis of Ribosome and Initiation Factor Dynamics during the Late Stages of Translation Initiation

Protein synthesis in all organisms is catalyzed by a highly-conserved ribonucleoprotein macromolecular machine known as the ribosome. Prior to each round of protein synthesis in the cell, a functional ribosomal complex is assembled from its component parts at the start site of a messenger RNA (mRNA) template during the process of translation initiation. In bacteria, rapid and high-fidelity translation initiation is promoted by three canonical initiation factors: IF1, IF2, and IF3. In this thesis, I report the use of single-molecule fluorescence methods to study the role of the initiation factors and ribosome-factor interactions in regulating molecular events that occur during late stages of the translation initiation pathway. In Chapter 1, I provide a structural and biochemical framework for understanding one of the key events of the initiation pathway: docking of the large (50S) ribosomal subunit with the small subunit 30S initiation complex (30S IC). The 50S subunit joining reaction is catalyzed by GTP-bound IF2 and results in formation of a 70S initiation complex (70S IC) that contains an initiator transfer RNA (tRNA) and is primed for formation of the first peptide bond. During 50S subunit joining, IF2-GTP establishes interactions with RNA and protein components of the 50S subunit's GTPase-associated center (GAC), which play an important role in subunit recruitment as well as the subsequent activation of GTP hydrolysis by IF2. In Chapter 2, I describe the development of a single-molecule fluorescence resonance energy transfer (smFRET) signal to monitor the interactions between IF2 and the ribosome's GAC during real-time 50S subunit joining reactions. Specifically, the role of the L11 region, comprising ribosomal protein L11 and its associated ribosomal RNA (rRNA) helices, was investigated. The L11 region is a prominent structural component of the GAC that is believed to undergo large-scale conformational changes during protein synthesis; however, the nature and timescale of these conformational dynamics, and their role in regulating the biochemical activities of IF2 during initiation, are not known. I demonstrate that my smFRET-based 50S subunit joining assay is sensitive to conformational rearrangements between IF2 and L11 within the 70S IC and can thus be used as a tool for characterizing GAC dynamics and elucidating their function during initiation. Furthermore, my smFRET approach is shown to provide information on the rate of 50S subunit joining as well as the rate of IF2 dissociation from the 70S IC. Notably, IF2-dependent GTP hydrolysis was found to influence the extent of 70S IC conformational dynamics as well as the dissociation rate of IF2. The role of IF3 in regulating 50S-subunit joining dynamics is discussed in Chapter 3. IF3 plays an important role in ensuring the fidelity of translation initiation by preventing the formation of initiation complexes containing a non-initiator tRNA and/or a non-canonical mRNA start codon. Inclusion of IF3 within the 30S IC in the smFRET experiments was found to render the IF2-catalyzed 50S subunit joining reaction highly reversible. Direct observation of repetitive docking and undocking of the 50S subunit with the 30S IC indicates that IF3 may modulate translation initiation efficiency by influencing the stability of the 70S IC. The individual 50S subunit docking events were found to result in the formation of very different classes of 70S IC, characterized by different stabilities and unique patterns of IF2-L11 interactions. I propose that these dynamics reflect an underlying conformational equilibrium of the IF3-bound 30S IC that is read out during 50S subunit joining, and that this equilibrium could be modulated in order to regulate the efficiency of translation initiation. Following initiation-factor mediated assembly of the 70S IC, the first aminoacyl-tRNA is delivered to the ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP. Accommodation of aminoacyl-tRNA into the ribosome's peptidyl transferase center leads to formation of the first peptide bond, which signals the end of initiation and entry into the elongation phase of protein synthesis. The ternary complex binding site on the ribosome overlaps with that of IF2 at the GAC; a question of key mechanistic importance in understanding how the ribosome coordinates the transition from initiation to elongation thus concerns the relative timing of ternary complex binding with respect to IF2 dissociation from the 70S IC. In Chapter 4, I present preliminary results from two- and three-color fluorescence co-localization experiments aimed at characterizing the timing of these events at the single-molecule level. The data strongly suggest the occurrence of simultaneous occupancy of the ribosome by IF2 and ternary complex, implying that the ribosome is structurally capable of recruiting ternary complex prior to IF2 release from the 70S IC. The observation that the ribosome can accommodate more than one translation factor at a time may have important implications for understanding how it efficiently coordinates factor binding and release throughout protein synthesis, and opens the door to mechanistic studies of the ribosomal L7/L12 stalk, presumed to play a prominent role in these processes

Molecular target of enteric VapC toxins and regulation of vapBC transcription by conditional cooperativity

The uibiquitous Type II toxin – antitoxin (TA) loci encode two proteins, a toxin and an antitoxin. The antitoxin combines with and neutralizes a cognate toxin. Usually, the TA genes form an operon that is transcribed by a single promoter located upstream of the genes. In most cases, the antitoxin autoregulates the TA operon via binding to operator sites in the promoter region. In almost all such cases, the toxin act as a co-repressor of transcription as the toxin enhances the DNA binding of the antitoxin. Recently, it has been shown that toxins play an additional role in stimulating transcription, as the antitoxin and toxin ratio is important for cooperative binding of the complex to DNA. The antitoxin is rapidly degraded by cellular proteases under conditions of stress and treatment with antibiotics, which leads to activation of the toxin. The toxins of TAs belong to different gene families. The most abundant TA gene family is vapBC that, in some organisms, have expanded into cohorts of genes. For example, the major human pathogen Mycobacterium tuberculosis contains at least 88 TAs, 45 of which are vapBC loci. VapC toxins encoded by vapBC loci are PIN domain proteins (PilT N-terminal). Eukaryotic PIN domain proteins are site-specific ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. From in vitro experiments it has been postulated that VapC toxins are RNases or DNases but their exact cellular target has remained elusive. Here I show that VapC encoded by Shigella flexneri 2a virulence plasmid pMYSH6000 and the chromosome of Salmonella enterica serovar Typhimurium LT2 are site-specific endoribonucleases that specifically cleave tRNAfMet in the anticodon stem-loop in vivo and in vitro. Furthermore, I show that VapC dependent depletion of tRNAfMet leads to bacteriostatic inhibition of global translation, which surprisingly induces low-level initiation of translation at elongator codons that are correctly positioned relative to a Shine & Dalgarno sequence. I also show that VapC forms a complex with VapB and acts as a co-repressor of vapBC transcription. During steady state growth VapB is in excess of VapC. However, nutrient stress or treatment with antibiotics leads to Lon protease dependent decrease in VapB levels. Furthermore, I show that VapC in excess of VapB directly interferes with cooperative DNA binding of the VapBC complex, which is dependent on the dimerisation of the VapC toxin. 2 In conclusion, I show that enteric VapCs not only regulate global cellular translation by tRNAfMet cleavage, but also regulate vapBC transcription by conditional cooperativity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo