Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...

The global re-emergence of TB and other mycobacterial infections have underscored the need for a thorough investigation of the biology of the causative agent, Mycobacterium tuberculosis, at the molecular level. The peculiar features of the bacterium such as slow growth rate, dormancy, unique cell wall composition and resistance towards phagocytosis by macrophages demands a detailed understanding of different essential molecular processes including transcription in this genus. Sequencing of several mycobacterial genomes provided an impetus for understanding the gene function and regulation of this formidable pathogen. Transcriptional regulation is one of the major mechanisms controlling gene expression. While a number of transcription units, promoters, sigma factors, and gene functions were identified and characterized, key features of transcription process are yet to be understood. The current study aims to understand some of the facets of transcription initiation and elongation in mycobacteria. The thesis is divided into five chapters. Chapter 1 introduces the bacterial transcription process. It starts with the description of the central molecule in transcription -the RNA polymerase (RNAP) and its catalytic mechanism. In the next section, each step of the transcription initiation, elongation and termination has been discussed. The mechanistic details as well as the different cellular factors involved in the regulation of the transcription have been discussed. The final part gives an overview of the transcription machinery of the mycobacteria, describing the promoter specificity and regulation of different sigma factors and other transcription factors known till date in mycobacteria. The scope and the objectives of the thesis are presented at the end of this chapter. In Chapter 2, a method of purification of RNAP from mycobacteria for optimized promoter -polymerase interactions is described. In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNAP with high specific activity is necessary to carry out a variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low σA-promoter specificity in promoter -polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of σA required for house - keeping transcription. In vivo reconstitution of RNAP holoenzyme with σA and its purification procedure which resulted in a holoenzyme with stoichiometric σA content is described in this chapter. The reconstituted holoenzyme showed enhanced promoter -specific binding and transcription activity compared to the enzyme isolated using standard procedure. Chapter 3 is aimed at the comparison of promoter - specific events during transcription initiation in mycobacteria. DNA -protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNAP binds to promoters in a sequence -specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. The first detailed kinetic analysis of promoter - RNAP interactions during transcription initiation in the σA-dependent promoters PrrnAPCL1, PrrnB and Pgyr of M. smegmatis are presented in this chapter. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to the distinct kinetic patterns, each one of the house -keeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. Moving the focus of the thesis from transcription initiation to elongation, a transcript cleavage factor of M. tuberculosis has been characterized in Chapter 4. After initiation of transcription, a number of proteins participate during elongation and termination by modifying the properties of the RNAP. Gre proteins are one such class of transcription elongation factors which are conserved across bacteria. They regulate transcription by binding near the secondary channel of RNAP, projecting their N-terminal coiled-coil domain into the active center and stimulating hydrolysis of the newly synthesized RNA by RNAP in the backtracked elongation complexes. Rv1080c is a putative gre factor homolog (MtbGre) present in M. tuberculosis.The protein enhanced the efficiency of promoter clearance by lowering the abortive transcription and also rescued the arrested and paused elongation complexes efficiently in the GC rich mycobacterial template. The Gre factor of M. smegmatis encoded by the gene MSMEG_5263 also showed biochemical properties similar to the M. tuberculosis protein. Although the mycobacterial Gre is similar in domain organization and shared the key residues for catalysis and RNAP interaction with Escherichia coli Gre proteins, it could not complement the E. coli strain deficient in Gre factors. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein - protein interactions for transcript cleavage. Decrease in the level of MtbGre also reduced the bacterial survival by several fold indicating its essential role in mycobacteria and suggesting that a single Gre copes up with the burden of transcription fidelity of the genome. Chapter 5 describes the studies carried out to identify Gre factor homologs in mycobacteria and deciphering their function during transcription. Gre factors are members of a growing family of proteins which regulate RNAP through secondary channel. Apart from the Gre factor, putative members of this class of proteins are identified in both M. smegmatis and M. tuberculosis.The closest homologue of the canonical Gre factor of M. tuberculosis in its genome is Rv3788. The protein has Gre factor like domain organization and possess the key acidic residues required for transcript cleavage activity and the putative hydrophobic RNAP interacting residues in the C-terminus similar to MtbGre. Despite having these common features, Rv3788 did not stimulate transcript cleavage. In contrast, it turns out to be a transcription inhibitor by preventing the binding of NTPs to the enzyme. The transcription inhibition is not promoter specific, and is mediated by its binding to RNAP through the secondary channel with its N-terminus coiled coil domain. Like M. tuberculosis, the fast growing non-pathogenic mycobacteria M. smegmatis also has an ORF (MSMEG_6292) which is homologous to its canonical Gre factor and it interacts with RNAP in a similar manner. However, this protein did not exert any transcript cleavage or inhibitory activities but could compete with the Gre factor for binding to RNAP. The Gre factor homologs in mycobacteria may be involved in regulation by inhibiting transcription or by blocking the RNAP secondary channel from other RNAP active site modulators.

Submitted by Michele Mologni (mologni@unoeste.br) on 2017-06-23T19:31:58Z No. of bitstreams: 1 Luciana Machado Guaberto.pdf: 1335752 bytes, checksum: aa6bb850bf2257d472ca5dbfd2512a5a (MD5) / Made available in DSpace on 2017-06-23T19:31:58Z (GMT). No. of bitstreams: 1 Luciana Machado Guaberto.pdf: 1335752 bytes, checksum: aa6bb850bf2257d472ca5dbfd2512a5a (MD5) Previous issue date: 2016-12-16 / The DOF family proteins (DNA binding with One Finger) comprises unique plant transcription factors (FTs) characterized by the presence of a DNA binding domain Dof containing a similar structure to the "zinc-finger" domain. These transcription factors are involved in different roles in various biological processes in plants. Although the analysis and genomic characterization of this gene family was performed in many plant species, information on Dof genes in sweet orange (Citrus sinensis (L.) Osbeck) and their involvement in the development of the fruits is still limited. For the full identification of CsDof genes in C. sinensis, including the structures of genes, chromosomal locations, introns and phylogeny, the examination of three databases resulted in the identification of 24 genes of this FT family distributed on 7 out of 9 chromosomes of this species. Phylogenetic analysis and classification of Dof transcription factors in C. sinensis was compared with their orthologs of Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.), which allowed their classification in four major groups (A, B, C and D) and 9 subgroups (a, B1, B2, C1, C2.1, C2.2, C3, D1, D2) of DOF proteins. For gene expression analysis, the 12 Dof genes with higher abundance of transcripts in fruits based on public RNA-seq data were selected for further analysis by semi-quantitative RT-PCR. This analysis revealed the CsDof genes were differentially expressed at different stages of development (up to 90 days after anthesis). It was also possible to establish three groups regarding their transcriptional activity relative to that in leaf tissue: genes with high (up to 8X), intermediate (up to 3X) and low relative expression (below 3 X). The gene CsDof1 showed higher expression in all of the fruit development stages, indicating that this isoform plays an important role in regulating the development of the fruits of C. sinensis. Taken together, our results provide new information about the regulation of Dof genes in controlling the formation of fruits of this important fruit species. / A família de proteínas Dof (DNA binding with One Finger) compreende fatores de transcrição (FT) exclusivos de plantas, caracterizados pela presença do domínio Dof de ligação ao DNA Estes fatores de transcrição estão envolvidos em diversos processos biológicos em plantas. Embora a análise e caracterização genômica de genes desta família tenha sido realizada em muitas espécies, informações sobre genes Dof em Citrus sinensis (L.) Osbeck (laranja doce) e o seu envolvimento no desenvolvimento do fruto é limitado. Para a identificação completa dos genes CsDof em C. sinensis, incluindo as estruturas dos genes (Exons - Íntrons), localizações cromossômicas e filogenia, três bancos de dados foram analisados. Como resultado desta busca 24 genes desta família de FTs distribuídos em 7 dos 9 cromossomos desta espécie. A análise filogenética e classificação dos fatores de transcrição Dof em C. sinensis foi realizada com a inclusão dos seus ortólogos de Arabidopsis (Arabidopsis thaliana L.) e arroz (Oryza sativa L.), sendo possível estabelecer quatro grupos principais (A, B, C e D) e 9 subgrupos (A, B1, B2, C1, C2.1, C2.2, C3, D1 e D2) de proteínas Dof. Foram selecionados 12 genes putativamente mais expressos em frutos de acordo com dados públicos de RNA-seq para a análise da expressão gênica por RT-PCR. Estes genes foram diferencialmente expressos durante as fases iniciais do desenvolvimento do fruto, sendo possível estabelecer três grupos: genes com alta expressão (acima de 8 X), expressão intermediária (acima de 3 X) e baixa expressão relativa (abaixo de 3 X) em relação à atividade transcricional em folhas. O gene CsDof1 se destacou como o gene com a maior expressão, sendo que esta manteve-se elevada em todos os estádios de desenvolvimento do fruto, evidenciando que esta isoforma desempenha um importante papel na regulação do desenvolvimento de frutos de C. sinensis. Considerados em conjunto, os nossos resultados fornecem novas informações sobre os genes CsDof na complexa rede regulatória envolvida no desenvolvimento dos frutos de laranja doce.

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In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.

Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity. Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor. Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress. Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function. Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.

Understanding the molecular structure of DNA was considered as greatest achievement in modern biology. It helped in understanding fundamental cellular processes such as replication of DNA, nature of the genetic code and transcription. It also led to technological advancements such as DNA sequencing, genetic engineering and gene cloning. The DNA molecule is highly polymorphic in nature and its structure is dependent on environment, base composition and sequence context. B-DNA, A-DNA, Z-DNA and curved or kinked DNA are some of the well characterized double helical polymorphs. B-DNA is the most prevalent structure in vivo and it can undergo small local variations and global variations. In this thesis we refer to distinct structural property of any particular DNA sequence as deviation from fibre model B-DNA structural parameters or random sequence DNA. Structural properties of DNA are an outcome of the linear arrangement of the 4 chemically different nucleotide bases and the characteristic features of the two grooves (minor and major) arising due to the asymmetric position of glycosidic bonds of base pairs. DNA structure and properties are expected to vary along its length. Several structural features have been defined for DNA duplex, while DNA stability, bendability and intrinsic curvature are well studied and found to be biologically relevant. These three sequence dependent properties differ in their nature and information content and can be studied both at local and global levels, depending on the length of DNA fragment being examined. Majority of the work in this thesis focuses on the analysis of these three DNA structural features in promoter regions of different eukaryotic systems and their relationship with gene expression. The thesis work is divided in to five sections briefly described below. The sections discuss prevalence of the three structural features, DNA stability, bendability and intrinsic curvature in the promoter regions of six eukaryotic systems namely S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human. The relationship between DNA structural features of promoter regions of S. cerevisiae with gene expression variability is discussed, followed by application of the structure-based promoter prediction algorithm ‘PromPredict’ in annotating promoter regions of six different eukaryotes. Finally, an analysis of structural features of the flanking sequences of transcription factor binding sites (TFBSs) of six transcription factors and their relationship with the DNA binding affinity is discussed. Each of the projects described below will appear as a separate chapters in the thesis. An overview of the eukaryotic transcription machinery, promoter elements and different DNA structural properties are discussed in the introduction of the thesis (chapter 1). The structural properties of DNA in the promoter regions of eukaryotic genes (chapter 2)Earlier studies in the lab reported that, apart from sequence motifs, promoter re- gions have distinct structural properties, such as lower stability, lesser bendability and more curvature compared to other genomic regions. But those studies were on small datasets and few model systems. Advancement in high-throughput tech- niques has made availability of transcription start site information for many model systems. This work was initiated with the aim of investigating the structural fea- tures in different eukaryotic systems belonging to different domains of life. The quantitative analysis of three different structural features of promoter regions of six different model systems S. cerevisiae, C. elegans, D. melanogaster, zebrafish, mouse and human has been carried out. Further, the composition of different k-mers (k=3, 4 and 6) A-tracts and G-quadruplexes has been studied. The analysis allowed us to understand the similarities and differences in struc- tural features of promoter sequences in different model systems. The core promoter sequences of S. cerevisiae, C. elegans, D. melanogaster, zebra fish, mouse and hu- man have been observed to be less stable and have lower preference for nucleosome formation. S. cerevisiae, C. elegans and D. melanogaster promoter sequences have been shown to be less bendable whereas zebrafish, mouse and human promoter se- quences are flexible in terms of bendability towards major groove as predicted fDNase 1 sensitivity model. S. cerevisiae, C. elegans, D. melanogaster core promoter regions have AT rich oligomers, whereas mouse and human core promoter regions have GC rich oligomers and G-quadruplex motifs. DNA structural features of TATA-containing andTATA-less promoters (chapter 3)Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA-box in their promoter sequences. Experiments on both classes of genes have reported that, they have differences in regulation of gene ex- pression and cellular functions. In this chapter, the differences in compositional and structural features of TATA-containing and TATA-less promoters in the above mentioned model systems are discussed. The results suggested that DNA structural features of TATA-containing and TATA-less promoters are distinctly different in all eukaryotes. The TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters in lower eukaryotes. In mouse and hu- man genes, DNA duplex stability and G-quadruplex motifs are very distinguishing features in the two classes of promoters. DNA structural properties of eukaryotic promoter regions and gene expression variability (chapter 4) Gene expression is regulated by various external (environment and evolution) and internal (genetic) factors. Presence of sequence motifs, such as TFBSs and TATA- box, as well as DNA methylation has been implicated in the regulation of expression of some genes in vertebrates, but a large number of genes lack these sequences. Ear- lier analyses (described in previous sections) in S. cerevisiae, have shown that their promoter sequences have special structural properties, such as low stability, less bendability and more curvature compared to other genomic regions. These strutural features may play a role in transcription initiation and regulation of gene expression. This project was carried out to understand 1. What is the relationship between DNA structural features and gene expres- sion? 2. What is the relationship between gene expression and bidirectionality of a pro- moter region? For this purpose, the information of seven different gene expression variability measures, stochastic noise, responsiveness, stress response, trans variability, mu- tational variance, interstrain variation and expression divergence have been com- pared with structural features in the promoter regions. It is observed that a few of the variability measures of gene expression are linked to DNA structural prop- erties, along with nucleosome occupancy, TATA-box presence and bidirectionality of promoter regions. Interestingly, gene responsiveness is shown to be most, inti- mately correlated with DNA structural features and promoter architecture. The study highlights the importance of sequence dependent structural features in gene regulation. Promoter prediction in eukaryotes using DNA duplex stability (chapter 5) Structural property-based algorithms can discriminate promoter sequences from non-promoter sequences and are far better than sequence motif-based predictors. Compared to other structural features, low stability is found to be the most preva- lent feature in promoter regions. “PromPredict” (in-house algorithm) uses the din- ucleotide free energy values obtained from differential melting stability of DNA du- plexes as a predictor of promoters and has been successfully used earlier to annotate promoter sequences in prokaryotes and rice. Comprehensive analysis of the perfor- mance of PromPredict in S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human as well as TATA-containing and TATA-less promoter regions of S. cere visiae with TSS data and 48 eukaryotic systems with translation start site (TLS) data revealed that differential stability is a good criterion for promoter prediction. DNA structure in flanking sequences of consensus motifs modulate transcription factor binding (chapter 6) Sequence specific DNA-protein interactions are essential for specific expression pat- terns during the development. There are several factors contribute to DNA-binding specificities of transcription factors (TFs). They include structure and flexibility of TFs, cofactors, chromatin environment and DNA sequence. Along with actual tran- scription factor binding sites (TFBSs), their sequence context (flanking sequences) is also shown to play a major role in gene regulation. Most of the studies have ad- dressed the sequence context at global level but very little is understood about the role of sequences flanking TFBSs in binding of transcription factors. This project was initiated with the aim of understanding the effect of flanking sequences of TFBSs in transcription factor binding affinity. In vitro DNA binding information of six different transcription factors (with three types of DNA bind- ing domains, Zinc finger (GATA4), home domain (AbdA, AbdB and Ubx) and bZIP (fos-jun and Nfil3)) was provided by Aseem Ansari’s lab. The compositional and structural features (minor groove width, propeller twist, wedge and free energy) are compared with the DNA binding profiles of 12mers (or 8mers) of six different transcription factors. It has been observed that some of the DNA structural proper- ties of flanking sequences are strongly correlated with binding affinity. For GATA4 sequences, binding affinity is negatively correlated to GC content or minor groove width at their 5′ -flanking region, showing the significance of narrow minor groove at 5′ -region. On the other hand, the binding affinity of bZIP proteins is negatively correlated to wedge angles, whereas in case of homeodomain proteins, it is posi- tively correlated to propeller twist and GC content. Thus, this study highlights the differential preference for flanking sequences outside the core binding motifs of six different TFs, which interact with DNA through α-helix. ‘The relationship between transcription pre-initiation complexes and gene ex- pression variability in S. cerevisiae’ is briefly described in the appendix section of the thesis. General conclusion Overall, the results presented in this thesis indicate that DNA sequence based structural features are unique to promoter regions and play an important role in gene regulation. Local structural features of flanking sequences of transcription factor binding sites are also instrumental in determining the DNA binding affinity of transcription factors.

Magister Scientiae - MSc / Neurological diseases are socially disabling and often mortal. To efficiently combat these diseases, a deep understanding of involved cellular processes, gene functions and anatomy is required. However, differential regulation of genes across anatomy is not sufficiently well understood. This study utilized large-scale gene expression data to define the regulatory networks of genes expressing in the hippocampus to which multiple disease pathologies may be associated. Specific aims were: ident i fy key regulatory transcription factors (TFs) responsible for observed gene expression patterns, reconstruct transcription regulatory networks, and prioritize likely TFs responsible for anatomically restricted gene expression. Most of the analysis was restricted to the CA3 sub-region of Ammon’s horn within the hippocampus. We identified 155 core genes expressing throughout the CA3 sub-region and predicted corresponding TF binding site (TFBS) distributions. Our analysis shows plausible transcription regulatory networks for twelve clusters of co-expressed genes. We demonstrate the validity of the predictions by re-clustering genes based on TFBS distributions and found that genes tend to be correctly assigned to groups of previously identified co-expressing genes with sensitivity of 67.74% and positive predictive value of 100%. Taken together, this study represents one of the first to merge anatomical architecture, expression profiles and transcription regulatory potential on such a large scale in hippocampal sub-anatomy. / South Africa

RNA polymerase binding protein A (RbpA) was first discovered as a RNA polymerase binding protein from Streptomyces. coelicolor. It was shown to cause rifampicin tolerance to RNA polymerase in vitro and leads to basal level of rifampicin resistance in vivo. This protein is exclusively present in the actinobacteria family with the nearest neighbour in mycobacteria. When null mutant of RbpA in S. coelicolor were transformed with the rbpA gene from Mycobacterium tuberculosis the resistance level of rifampicin increased from 0.75 µgml-1 to 2 µg ml-1 suggesting analogous role of MtbRbpA (RbpA from M. tuberculosis). MsRbpA, RbpA from Mycobacterium smegmatis was found to interact with the β-subunit of RNAP and its binding location on M. smegmatis RNAP was shown to be 18 Å from the (i+1) site. MsRbpA was also shown to rescue the inhibitory effect of rifampicin in vitro. Furthermore, overexpression of MsRbpA in wild type M. smegmatis resulted in the increase in the MIC of rifampicin to 85 µg ml-1 from 20 µg ml-1, which is the MIC of rifampicin for the wild type M. smegmatis. On the other hand, MsRbpA was unable to augment transcription in the presence of rifampicin when the reaction was catalysed by rifampicin resistant RNAP. Recent reports have shown that MtbRbpA enhances the affinity σA to core RNAP thereby activates transcription. The N and C-termini of MtbRbpA interact with σA while the C-terminal region of MtbRbpA is required for the oligomerisation of MtbRbpA. However M. tuberculosis and S. coleicolor are part of same family actinobacteria, RbpA is essential for the former while it is dispensable in the later case.This work focuses on characterisation of rifampicin resistant RNAP from M. smegmatis and elaborates on the roles played by MsRbpA. These include its effect on transcription activation, transcription rescue, its role in RNAP promoter closed and open complex formation, characterisation of its site of interaction with RNAP and σA, finding critical functional residues and establishing the essentiality of MsRbpA in M. smegmatis. Chapter 1 deals with the literature survey on structure of bacterial RNAP, promoters, sigma factors, RNAP inhibitors, transcriptional activators with the emphasis on the Mycobacteria. Chapter 2 summarises the identification of the mutations in rpoB gene from the rifampicin resistant (RifR) mutant strains of M. smegmatis, purification of RNAP from these strain, determining IC50 values of these RifR RNAP for rifampicin, finding kinetic parameters for the interaction of RifR RNAP with 3-formyl rifampicin and evaluating their interaction with MsRbpA. Chapter 3 describes the function of MsRbpA in transcription initiation, particularly its role in RNAP-promoter closed and open complex formation. Furthermore, this chapter throws light on the role of MsRbpA in transcription activation vis a vis its effects on transcription rescue from the inhibitory effect of rifampicin. Chapter 4 elucidates the function of a segment of MsRbpA from Arg58 to Lys 73 in activation of transcription activity, transcription rescue from the inhibitory effect of rifampicin and its interaction with σA and core RNAP. Furthermore, the alanine scanning of the region and subsequent in vitro transcription studies revealed four important residues required for MsRbpA functions. Chapter 5 describes the generation of conditional knock down strain of MsRbpA in M. smegmatis and establishing its essentiality. Chapter 6 summarizes the work documented in the thesis.

Le TNF-α est une cytokine pro-inflammatoire du système immunitaire qui, lorsque sa production est déréglée, induit de nombreuses pathologies chez l’homme (cachexie, arthrite rhumatoïde, etc.) Outre une régulation transcriptionnelle de cette cytokine, il existe aussi une régulation post-transcriptionnelle permettant un contrôle affiné de sa production. Le laboratoire de Biologie du Gène étudie cette régulation post-transcriptionnelle faisant intervenir une séquence consensus dans l’ARNm appelée séquence AU-riche (ou ARE pour AU-rich element) et les protéines qui y sont impliquées. Généralement, les ARNm porteurs d’ARE codent pour des protéines dont l’expression est transitoire. Ces gènes requièrent un contrôle très précis de leur expression et c’est pourquoi, en plus d’être soumis à de nombreux contrôles transcriptionnels, la traduction et la stabilité de leurs ARNm sont très finement régulées. La réponse immune innée implique de nombreux ARNm de ce type. Jusqu’à présent, la fonction de la protéine TIAR dans la régulation de l’expression du TNF-α n’a pas été complètement élucidée. Outre le TNF-α, la participation à la réponse immune innée de nombreuses protéines encodées par des ARNm porteurs d’ARE pourrait conférer à la protéine TIAR un rôle de régulateur essentiel dans le contrôle de l’inflammation. Nous avons donc générés plusieurs lignées de macrophages RAW 264.7 surexprimant la protéine TIAR entière ou différents mutants de TIAR afin de déterminer, par une analyse globale par puces à ADN, les ARNm cibles de TIAR au cours de la réponse immune. Cette approche nous a permis de démontrer que la protéine TIAR exerce un contrôle sur le métabolisme de l’ARNm du TNF-α et de MKP-1 (MAP kinase phosphatase 1), une phosphatase majeure dans la voie de signalisation de la MAPK p38. Cette voie de signalisation joue un rôle essentiel dans la stabilisation et la traduction de nombreux ARNm porteurs d’ARE encodant des protéines de la réponse inflammatoire. D’autre part, nous avons voulu étudier in vivo la fonction de la protéine TIAR au cours de la réponse immune. Nous avons, dans ce but, généré des souris transgéniques surexprimant l’isoforme courte de la protéine TIAR. Si nous n’avons pas encore pu mesurer les effets d’une surexpression de TIAR sur la réponse inflammatoire chez ces souris, ces individus transgéniques ont révélé qu’une expression anormale de la protéine TIAR induit une létalité importante au cours du développement embryonnaire. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished