No description available.

Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell.   To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize.  In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Il est maintenant clairement établi que la biogenèse des ribosomes est l’un des nombreux processus cellulaires qui voit sa régulation profondément modifiée au cours de la transformation cellulaire.Toutefois, si il est bien décrit que le taux synthèse des ribosomes est augmenté au cours du processus tumoral, de plus en plus de données suggèrent que les étapes posttranscriptionnelles peuvent aussi être altérées. Dans ce contexte biologique, les objectifs de cette thèse sont de déterminer si : i) la maturation de l’ARNr est altérée en plus de l’augmentation de sa synthèse ; ii) cette altération peut conduire à la synthèse de ribosomes modifiés dont la fonction est altérée ; iii) ces modifications participent directement à la dérégulation traductionnelle observée dans les cellules cancéreuses. Pour cela, nous avons étudié les principales étapes de la biogenèse des ribosomes ainsi que la composition des ribosomes et leurs capacités fonctionnelles dans différents modèles de progression et/ou d’agressivité tumorale. Les résultats obtenus montrent qu’en plus de l’augmentation du taux de synthèse des ribosomes, les étapes post-transcriptionnelles sont modifiées, en particuliers le niveau de méthylation de l’ARNr. Ces modifications sont associées à des défauts importants de traduction (saut de codon stop, incorporation erronée des acide-amines) et particulièrement à une augmentation de la traduction IRES-dépendante de facteur clefs de la tumorigénèse. Dans leur ensemble, ces résultats suggèrent que les modifications de la biogenèse des ribosomes pourraient être une étape clef de la cancérogenèse, en modifiant les capacités traductionnelles des ribosomes cytoplasmiques / Ribosome biogenesis is a fundamental and extremely complex cell process. In mammals, ribosome synthesis coordinates the assembly of 80 proteins and 4 rRNA to form the two ribosomal sub-units. The maturation of the ribosome is a multi-step post-transcriptional process essential to obtain functional ribosomes. It is now well demonstrated that ribosome biogenesis and its regulation is altered during transformation process. However, if the increase of ribosome synthesis in cancer cell is well documented, there are numerous recent data suggesting that post-transcriptional steps could also be altered. In this biological context, the objectives of my Ph.D were to determine if: i) the maturation of rRNA is altered during the increase of ribosome synthesis; ii) these alterations could modify the ribosomes and alter their function and iii) these modifications directly participate to the deregulation of translation observed in cancer cells. We have explored the major steps of ribosome biogenesis as well as the structure of the cytoplasmic ribosomes and their functional capacity in different cellular models of tumor progression and/or aggressively. The results obtained show that in addition of the increase of the level of ribosome synthesis, post-transcriptional modifications are altered, particularly the level of rRNA methylation. These modifications are associated with strong defect of translation (stop codon bypass, misincorporation of amino-acid) and an increase of the IRES-dependant translation of important factors playing a crucial role in tumorigenesis. These results suggest that modifications of ribosome biogenesis could be a key step of cancer cell transformation

In this project a number of peptidyl transferase antibiotics were studied, specifically a group of aminohexose cytosine nucleoside antibiotics and their interaction with a selected number of highly conserved ribonucleic acid (RNA) motifs, designed to represent their possible binding site within the ribosome. This group of antibiotics shows a wide range of interesting properties, including antiviral and anti-tumour activity, and as they bind to a particularly conserved region in the ribosome, they are likely to be difficult for microorganisms to develop resistance to. It is hoped that once the mechanism of action of these antibiotics is better understood, that modifications to the antibiotics can be effectively made to create new or hybrid antibiotics with more selective antibacterial, or indeed antiviral or anti-tumour properties. The nuclear magnetic resonance (NMR) structure of the RNA binding, peptidyl tranferase inhibitor antibiotics amicetin, blasticidin S and gougerotin, in their native solution states, have been successfully determined. The structures all exhibit a stable conformation, stabilised by intramolecular hydrogen bonds. Amicetin was observed to be folded, distinctly different from the linear, extended conformation of amicetin previously determined by X-ray crystallography. The structure of blasticidin S was found to be very similar to its X-ray crystal structure. Gougerotin was shown to form a similar conformation to blasticidin S, save that the end chain of gougerotin was bent at right angles to the rest of the molecule, forming a structure similar to that of the major bound X-ray crystal structure of blasticidin S. All the solution structures showed a similar conformation in the analogous regions of their chemical structure, suggesting that hybrid antibiotics could be produced.Two highly conserved RNA motifs of Halobacterium halobium (H. h.) and Escherichia coli (E. coli) 23S ribosomal RNAs were chosen to investigate their interaction with amicetin. The NMR structure of the H. h. and E. coli. 29-mer RNA motifs have been determined; the motifs both form well folded A-form RNA conformations. The E. coli NMR structure differs from the X-ray crystal structure of the motif contained within the ribosome, as a highly conserved adenine residue, which resides in a bulge strongly implicated with amicetin binding, folds into the helix as opposed to being flipped out. Instead, an adjacent cytosine residue partially flips out; whereas in the crystal structure, it is folded within the helix. The NMR stuctures of the H. h. motif differs from the X-ray crystal structure of the motif, contained within the ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. To continue this study, a fully 13C and 15N isotopically labelled version of the H. h. RNA sample has been partially assigned, and an initial structure determination has been performed, using ultra high field 1 GHz spectroscopy.Addition of amicetin to both the H. h. and E. coli 29-mer RNA samples were accompanied by discrete changes to the spectra, suggesting weak interaction between the two components. These can be qualitatively interpreted to changes induced in the local conformation of the RNA motifs and the amicetin arising from the formation of a complex, between the amicetin and the bulge region of the particular motif.

La RNase E chez Escherichia coli est une endoribonucléase essentielle qui joue un rôle important dans la maturation des ARN stables, dans le contrôle qualité des ribosomes, ainsi que dans la dégradation constitutive et régulée des ARN messagers. La séquence de ciblage à la membrane (MTS pour Membrane Targeting Sequence), qui forme une hélice α-amphipatique, ancre la RNase E à la membrane cytoplasmique interne des cellules. La conservation absolue du MTS chez l'ensemble des -protéobactéries suggère un rôle important de la localisation membranaire RNase E dans le métabolisme de l'ARN. Pour élucider la fonction cellulaire de l'association membranaire de la RNase E, nous avons caractérisé la souche rne∆MTS qui exprime une RNase E cytoplasmique. Les résultats de cette étude nous amènent à proposer que l'association membranaire de la RNase E est nécessaire à la stabilité de la RNase E, est impliquée dans des interactions fonctionnelles avec des régulateurs associés à la membrane et protège les transcrits présents dans le nucléoïde en évitant des interactions prématurées avec la RNase E. En particulier, garder la RNase E à la membrane est critique pour la spécificité de la RNase E dans le contrôle qualité des ribosomes. Cette association membranaire est une nouvelle couche de régulation qui permet d’expliquer comment la RNase E, une enzyme avec peu de spécificité de séquence et avec beaucoup de substrat, peut remplir les fonctions de «maturase» et de «dégradase». / RNase E in Escherichia coli is an essential endoribonuclease with important roles in stable RNA maturation, in ribosome quality control and in constitutive and regulated mRNA degradation. The Membrane Targeting Sequence (MTS), which forms an amphipathic α-helix, anchors RNase E on the inner cytoplasmic membrane. The absolute conservation of the MTS among -Proteobacteria suggests an important role for RNase E membrane association in RNA metabolism. To elucidate the cellular function of the membrane association of RNase E, we characterized the rne∆MTS strain expressing cytoplasmic RNase E. The results of this study lead us to propose that RNase E membrane association is necessary for RNase E stability, for functional interactions with membrane-associated regulatory factors and for protecting nascent transcripts in the nucleoid from premature interactions with RNase E. In particular, keeping RNase E to the membrane is critical for the specificity of RNase E in ribosome quality control. Membrane association is a new layer of regulation that can explain how RNase E, an enzyme with little sequence specificity and many substrates, can fulfill both ‘maturase’ and ‘degradase’ functions.

<p><a>Internal ribosome entry sites (IRES) are segments of the mRNA found in untranslated regions, which can recruit the ribosome and initiate translation independently of the more widely used 5’ cap dependent translation initiation mechanism. IRES play an important role in conditions where has been 5’ cap dependent translation initiation blocked or repressed. They have been found to play important roles in viral infection, cellular apoptosis, and response to other external stimuli. It has been suggested that about 10% of mRNAs, both viral and cellular, can utilize IRES. But due to the limitations of IRES bicistronic assay, which is a gold standard for identifying IRES, relatively few IRES have been definitively described and functionally validated compared to the potential overall population. Viral and cellular IRES may be mechanistically different, but this is difficult to analyze because the mechanistic differences are still not very clearly defined. Identifying additional IRES is an important step towards better understanding IRES mechanisms. Development of a new bioinformatics tool that can accurately predict IRES from sequence would be a significant step forward in identifying IRES-based regulation, and in elucidating IRES mechanism. This dissertation systematically studies the features which can distinguish IRES from nonIRES sequences. Sequence features such as kmer words, and structural features such as predicted MFE of folding, Q_MFE, and sequence/structure triplets are evaluated as possible discriminative features. Those potential features incorporated into an IRES classifier based on XGBboost, a machine learning model, to classify novel sequences as belong to IRES or nonIRES groups. The XGBoost model performs better than previous predictors, with higher accuracy and lower computational time. The number of features in the model has been greatly reduced, compared to previous predictors, by adding global kmer and structural features. The trained XGBoost model has been implemented as the first high-throughput bioinformatics tool for IRES prediction, IRESpy. This website provides a public tool for all IRES researchers and can be used in other genomics applications such as gene annotation and analysis of differential gene expression.</a></p>

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated ribosomal RNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We find that deletion of A1150 in the 16S rRNA blocks tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowers tagging levels at Pro-opal and rare Arg codons but not at the 3'-end of an mRNA lacking a stop codon. We conclude that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.