Structural And Biophysical Analysis Of The Regulatory Mechanism Of Mycobacterium Tuberculosis Sigma Factors
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.
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