Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Les expériences de séquençage à haut débit ont permis de démontrer que la transcription ne se limite pas aux régions codantes et qu’une grande partie du génome est transcrite en ARN non-codants (ARNnc). Parmi eux, les transcrits cryptiques sont initiés à l’intérieur des régions codantes. Des études faites chez la levure Saccharomyces cerevisiae, ont pu identifier plusieurs facteurs qui répriment la transcription cryptique. Un de ces facteurs est Spt6, une chaperonne d’histones requise pour le maintien d’un bon niveau de nucléosomes le long des gènes transcrits. Lorsque Spt6 est muté, on observe une déplétion des nucléosomes conduisant à l’activation des promoteurs cryptiques. Cependant, le mécanisme par lequel ces transcrits cryptiques sont régulés n’est pas encore clair. Dans ce mémoire, nous présentons un travail dans lequel nous avons développé une méthode probabiliste dans le but de caractériser les transcrits cryptiques à partir de données de RNA-Seq. Cette méthode est basée sur une cumulation des données et permet de tenir compte des variations dans l’expression et dans la longueur des gènes, grâce à une étape de randomisation des données. Les résultats démontrent que notre méthode est au moins aussi efficace que les méthodes précédemment décrites dans la littérature et offre un bon compromis entre le taux de faux positifs et de faux négatifs. Enfin, le plus important est que cette méthode permet de prédire les régions génomiques où les transcrits cryptiques sont initiés. Nous avons mis en évidence la présence de transcrits cryptiques sur les brins sens et antisens par rapport au gène. Nous avons également montré que les promoteurs cryptiques sens et antisens sont enrichis en motif TATA et que les transcrits cryptiques sont polyadénylés, ce qui suggère qu’ils peuvent être régulés par les mêmes mécanismes qui régulent les gènes. Alors que les transcrits cryptiques sur le brin sens se terminent à la même position que les gènes dont ils sont issus, les transcrits cryptiques sur le brin antisens terminent préférablement aux extrémités 3’ des gènes situés en amont. Nous proposons donc que les terminateurs chez S. cerevisiae ont évolué pour terminer la transcription de manière bidirectionnelle afin d’empêcher une transcription aberrante qui pourrait envahir les gènes voisins. / High throughout sequencing experiments have shown that transcription in not limited to coding regions and that most of the genome is transcribed into non-coding RNA (ncRNA). Among them, cryptic transcripts are aberrantly initiated from within the coding regions. Several studies in Saccharomyces cerevisiae have identified many factors that suppress cryptic transcription. One such factor is Spt6, a histone chaperone required for maintaining appropriate nucleosome levels on transcribed genes. In Spt6 mutant cells, nucleosomes are depleted, leading to activation of cryptic promoters. However, the mechanism by which these cryptic transcripts are regulated remains unclear. In this thesis, we present the development of a probabilistic method for the characterization of cryptic transcripts from RNA-Seq data. The method is used to characterize cryptic transcription in spt6-1004 cells. The method is based on a cumulative distribution function, thus taking into account variations in gene expression and gene length thanks to a data randomization step. Results show that our method is at least as good as previously published methods and provides a good compromise between false positives and false negatives. Importantly, this method allows for the prediction of genomic regions where cryptic transcripts are initiated. We have demonstrated the presence of cryptic transcripts running on the sense and antisense strands relative to genes. We also showed that, both sense and antisense cryptic promoters are enriched for TATA-like sequences and that cryptic transcripts are polyadenylated, suggesting that they may be regulated by the same mechanism that occurs on genes. While the cryptic transcripts on the sense strand terminate at the same position as the genes from which they are derived, cryptic transcripts on the antisense strand preferentially terminate at the 3’-end of upstream genes. We therefore propose that S. cerevisiae terminators have evolved to terminate transcription bidirectionally in order to prevent an aberrant transcription that could invade neighboring genes.

Doctor of Philosophy / Department of Grain Science and Industry / Finlay I. MacRitchie / Novel genetic materials were used to deduce gluten protein composition-functionality relationships. The Pegaso bread wheat near-isogenic lines (NILs) included addition, variation and/or deletion of major loci coding for HMW-GS, LMW-GS and gliadins. The waxy wheat lines (Svevo and N11 set) included wild, partial and complete waxy lines. Triticale translocations include 1R.1D and 1A.1D lines (GDS7, Trim, Rhino and Rigel sets) with HMW-GS 5+10 and 2+12. The main goal of the study was to establish the usefulness of NILs as appropriate materials to investigate the structure-function relationships of wheat proteins and to evaluate the performance of unique triticale translocations and waxy wheat lines. Effect of genetic variation on phytochemical (phenolic acid and policosanol) contents was also studied. Innovative methods like MALLS, Lab-on-a-chip and micro (10 g) baking were utilized along with traditional analytical methods. Results confirmed the potential of using NILs in understanding the effects of certain proteins coded at specific loci that might often be targeted in breeding programs. Removal of expected chain terminators at Gli-1/Gli-2 loci causes a shift in MWD to higher values, reflected in higher UPP and dough strength. Lines with HMW-GS 5+10 were clearly separated from 2+12 lines in terms of dough strength and UPP. The present study obtained evidence that modified ω-gliadins acts as chain terminators and cause reduction of protein polymer size and thus shifts in MWD. Marked differences in terms of milling characteristics, protein composition and ultimately in end-use functionality were observed with various waxy wheat null lines. Loaf volumes with waxy wheat flour alone were higher than a 50% blend with commercial wheat; however, breads were unacceptable to consumers in all aspects. Poor milling quality, very low mixing times with low bread loaf volumes were typical of all the triticales studied. However, translocation of the HMW-GS from wheat chromosome 1D increased dough strength, particularly the HMW-GS 5+10. Among the phytochemicals studied, double nulls at Gli-1 loci of Pegaso NILs had the highest total policosanols and total phenolic acid contents.Slight variation to wheat phenolic acid composition and contents were observed with waxy wheat and triticale lines.