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.

Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human pathogen, Mycobacterium tuberculosis, especially due to the ability of this bacterium to survive in the host, often for several decades prior to the onset of the disease. Transcription initiation factors, also called σ factors in prokaryotes, are diverse in size and sensory/regulatory mechanisms. Indeed, the number of alternate σ factors vary substantially from six in E. coli to more than 118 in Plesiocystis pacifica. The large number of alternative σ factors has been suggested to be correlated with the diversity of micro-environments experienced by a bacterial cell. Studies on several prokaryotic σ factors reveal common features in these proteins that was not evident earlier due to poor sequence conservation. A central theme that emerges from these studies is that a minimalistic architecture of two domains can recognize promoter DNA and recruit the RNAP enzyme to initiate transcription. Additional domains are required when certain promoter elements are missing or to enable a specific, context dependent regulatory mechanism. The work reported in this thesis was influenced by previous studies in this laboratory and elsewhere on M. tuberculosis σ factors. While these studies revealed multiple features of transcription initiation, several aspects of this mechanism, including some classes of σ factors remain to be examined. The focus of this study was to examine an under-explored sub-group of σ factors, classified as the ECF41 sub-group. This sub-group has an additional domain at the Carboxy-terminus that has been hypothesised to influence σ factor activity. Towards this goal, M. tuberculosis σJ was examined. Previous studies suggested a role for this σ factor in modulating the response to hydrogen peroxide stress. An intriguing feature based on sequence analysis was that neither did this extra-cytoplasmic function σ factor have an anti-σ factor that can respond to oxidative stress nor was it directly associated with a mechanism to sense oxidative stress. The specific goal of the research described here was to understand the structural and mechanistic features that govern σJ activity. This thesis is organized as follows- The first chapter provides a brief introduction to prokaryotic transcription and regulatory mechanisms that govern this process. This chapter also has the literature necessary to phrase the problem in characterizing this family of proteins with particular reference to the unique physiology of Mycobacterium tuberculosis. A summary of the previous work is provided in this chapter to place the current study in context of previous studies and highlight the lacunae in our understanding of the transcription mechanism in M. tuberculosis. Chapter two describes the structural characterization of M. tuberculosis σJ by single-crystal X-ray diffraction. The poor sequence similarity of σJ to known σ factors precluded efforts to obtain phase information by molecular replacement methods. Here we also describe the steps that were essential to obtain diffraction quality crystals and the subsequent steps to account for pseudo-merohedral twinning, an imperfection that could have potentially been a limitation for structure determination. The crystal structure of σJ provide an example of successful phase determination with data collected on near-perfectly twinned crystals using single-wavelength anomalous dispersion. Chapter three describes computational efforts to understand the regulatory mechanisms of M. tuberculosis σJ. Classical Molecular Dynamics (MD) simulations were performed to understand the role of a C-terminal SnoaL_2 domain in this transcription factor. The MD simulations suggest that the C-terminal SnoaL_2 domain limits inter-domain movements between σJ2 (the pribnow box binding domain) and σJ4 (the -35 promoter element binding domain) and confers a compact three domain organization to this protein. The biochemical and functional characterization of M. tuberculosis σJ is described in chapter four. This includes in vitro studies on σJ and cognate promoter DNA interactions performed using Surface Plasmon Resonance (SPR) and Electrophoretic Mobility Shift Assays (EMSA). The ex vivo reporter based experiments to examine the effect of SnoaL_2 domain on σJ activity are also described. Spectroscopic studies on σJ interactions with a small molecule limonene-1,2-epoxide suggested a potential novel role for the SnoaL_2 domain in σJ. Chapter five summarizes the work on M. tuberculosis σJ reported in this thesis. We note that this study opens up a new perspective to understand σ factors. In particular, M. tuberculosis σJ suggests that the domain organization is likely to be retained in ECF41 sub-group of σ factors. This study also hints at broader implications in the distinction between one-component systems and transcription factors. Bioinformatic analysis suggest that observations similar to that noted in M. tuberculosis σJ are likely to be more widespread across diverse phyla than currently acknowledged. This thesis has three annexures. Annexure-I summarizes experimental details of the work performed on the M. tuberculosis σ/anti-σ factor complex σH/RshA. Annexure-II summarizes experimental details and strategies that could not be incorporated in the main body of this thesis. Annexure-III describes a short project performed on a bi-domain protein tyrosine phosphatase PTP99A.