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In bacteria, the RNA polymerase (RNAP) consists of the following subunits: α2, β, β’, ω and σ. The core RNAP (α2ββ’ω) possesses the polymerising activity and it associates with one of the sigma factors to initiate transcription from a promoter region on the DNA template. All bacteria carry an essential housekeeping sigma factor and a number of extra cytoplasmic function (ECF) sigma factors. During alternate physiological states, a major part of transcriptional regulation is carried out by sigma factors, which act as transcriptional switches, thus, making it possible for bacteria to adapt to varied environmental signals by transcribing the necessary set of genes. Bacteriophages utilise various mechanisms for subverting the bacterial biochemical machinery for their advantage. One such example in E. coli is AsiA protein encoded by an early gene of T4 bacteriophage. Because of its property of binding to σ70, AsiA can inhibit transcription from E. coli promoters bearing –10 and –35 DNA sequences leading to inhibition of growth. σ70 of E. coli is also regulated by a stationary phase specific protein, Rsd, whose major function seems to be helping the cell in switching the transcription in favour of stationary phase genes. In this study we have investigated the mechanism of interaction of T4 AsiA and E. coli Rsd to σ70 of E. coli and also tried to determine the basis of differential inhibition of E. coli growth by AsiA and Rsd. In chapter one we have reviewed the published literature on regulation of transcription in bacteria. Some of the well known mechanisms of regulating gene expression are: DNA supercoiling, two component signal transduction system (TCS), regulation by alarmone ppGpp and 6S RNA, and sigma-antisigma interactions. Most bacteria carry a number of sigma factors and each of them is dedicated to transcribing genes in response to environmental signals. Intracellular levels of sigma factors and their binding affinity to core RNAP are deciding factors for initiating transcription from specific subsets of genes. In addition, sigma factor activity is also controlled by specific proteins, which bind to sigma factors (anti-sigma factors) under certain environmental conditions. A number of anti-sigma factors have been isolated from a variety of bacteria and the mechanisms of action of binding to cognate sigma factors have been worked out by using genetic, biochemical and structural tools. In chapter two, using yeast two hybrid assay (YTH), we have identified the regions of σ70 which interact with AsiA, and it was observed that amino acid residues from 547-603, encompassing region 4.1 and 4.2 are involved in binding to σ70. Interestingly, we found that truncated σ70 fragments lacking the N-terminal regions, apparently bound to AsiA with higher affinity compared to full length σ70. As AsiA expression, because of its transcription inhibitory activity, is inhibitory to E.coli growth, co-expression of the truncated C-terminal σ70 fragments (e.g. residues 493-613, σ70C121), which bind to σ70 with high affinity, could relieve growth inhibition. The complex of GST:AsiA-σ70C121 could be purified from E. coli cells. GST:AsiA purified from E .coli cells was found to be associated with RNAP subunits. Since further studies on this interaction required GST:AsiA preparation devoid of RNAP subunits, we decided to express this protein in S. cerevisiae. Bioinformatics analysis indicated the absence of a σ70 homologue in S.cerevisiae. As expected, GST:AsiA purified from the yeast was found to be free from any RNAP like proteins. The protein purified from yeast was used for in-vitro binding experiments. Our YTH analysis had indicated that deletion a part of region 4.1 or 4.2 of σ70 leads to loss of binding to AsiA. However, the published NMR structure of AsiA in complex with peptides corresponding to region 4 of σ70, showed that either region 4.1 or 4.2 alone can bind to AsiA indicating at the possible existence of two binding sites for AsiA. In order to confirm the physiological significance of this finding, we studied the interaction of truncated σ70 fragments lacking either region 4.1 or 4.2 with AsiA in-vivo in E. coli and in-vitro by affinity pull down assays. It was observed that σ70 fragments lacking either region 4.1 (σ70∆4.1) or 4.2 (σ70∆4.2), did not neutralize the GST:AsiA toxicity, indicating lack of interaction. The affinity purified GST:AsiA from these E. coli cells did not have σ70∆4.1 or σ70∆4.2 associated with it. Similar results were obtained from pull down assays in-vitro, where we found that σ70∆4.1 or σ70∆4.2 do not show any observable interaction with AsiA. This clearly established that the minimum region of σ70 required for physiologically relevant interaction with AsiA consists of both the regions 4.1 and 4.2. Chapter 3 of this thesis has been devoted to this aspect of AsiA-σ70 interaction. Having defined the minimum region of σ70 interacting with AsiA, we sought to identify the regions and amino acid residues of AsiA, which are critical for interaction with σ70. The approach for identification of mutants and their characterisation has been discussed in chapter 4. For this purpose, we made systematic deletions in the N and C-terminal regions of the protein and also isolated random mutants of AsiA, which lack binding to σ70 and thus are non-inhibitory to E. coli growth. It was found that deletion of 5 amino acids from N-terminus and 17 amino acids from C-terminus did not alter the inhibitory activity of AsiA. In contrast, deletion of N-terminal 10 amino residues led to complete loss of activity, while in the C-terminus, a gradual loss of activity was observed when amino acid residues beyond 17 amino acids were deleted. A 34 amino acids C-terminal deletion mutant was found to be completely inactive. E10K mutant was found to be inactive, but changes of E to other amino acids such as S, Y, L, A and Q were tolerated, indicating that negative charge at E10 is not a crucial element for interaction with σ70. Inactive mutants could be overexpressed in E. coli and showed reduced binding in YTH assay and were also poor inhibitors of in-vivo transcription in E. coli. We concluded that the primary σ70 binding site of AsiA is present in the N-terminus, yet C-terminal 64-73 amino acid residues are required for effective binding in-vivo. These studies also correlate the inhibitory potential of AsiA with its σ70 binding proficiency. In chapter 5, we have made a comparative analysis of mechanism of interaction of AsiA and Rsd to E. coli RNAP. Overexpression of Rsd was found to be less inhibitory to E. coli cell growth than that of AsiA. The affinity purified GST-AsiA from E. coli was found to have all the RNAP subunits associated with it, whereas, only σ70 was found to be associated with similarly purified GST:Rsd, pointing towards differences in binding to RNAP. In affinity pull down assays, in-vitro, it was found that both AsiA and Rsd do not show any observable binding to core RNAP. Binding of AsiA to σ70 in holo RNAP led to the formation of a ternary complex, whereas no ternary complex was observed when Rsd was made to interact with holo RNAP. Analysis of protein-protein interaction by YTH showed that region 4.1 and 4.2 are critical for binding of both AsiA and Rsd to σ70. However, in the case of Rsd, the surface of interaction is not limited to this region only and other regions of σ70 make significant contribution to this binding. Possibly, the interaction of Rsd with the core binding regions of σ70 prevents its association with core RNAP. Kinetic analysis of binding by surface plasmon resonance (SPR) showed that binding affinities (Kd) of AsiA and Rsd to σ70 are in similar range. Therefore, we concluded that the ability of AsiA to trap the holo RNAP is, probably, responsible for higher inhibitory activity of this protein compared to that of Rsd. Thus, T4 AsiA and E. coli Rsd, which share regions of interaction on σ70, have evolved differences in their mechanism of binding to RNAP such that T4 AsiA, by trapping the holo RNAP subverts the complete bacterial transcription machinery to transcribe its own genes. Rsd, on the other hand, has evolved to interact primarily with σ70, which favours the utilisation of core RNAP by other sigma factors.

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.

This PhD thesis is focused on the synthesis of DNA modified with photocleavable 2- nitrobenzyl protecting groups in major groove and its applications in the regulation of gene expression in the level of transcription. In the first part of my thesis, the synthesis of photocaged 2'-deoxyribonucleosides triphosphates and their photolysis to unprotected 5-hydroxymethylated nucleotides is described. All prepared nucleoside triphosphates were good substrates for their enzymatic incorporation into DNA. Synthesized 5-(2-nitrobenzyloxy)methyl-2'-deoxyuridine-5'- monophosphate (dUNBMP) and DNA with one 5-(2-nitrobenzyloxy)methyl- modification in the sequence were used for the detailed kinetic studies of photocleavage reactions. In the second part of the thesis, the series of modified DNAs with specific sequences were prepared by primer extension (PEX) and/or polymerase chain reaction (PCR). A cleavage of prepared modified DNAs was studied by selected restriction endonucleases (REs). In all cases, the nitrobenzylated DNA fully resist the cleavage by REs. The deprotection/ photocleavage conditions for nitrobenzylated DNA were studied in the case of DNAs with positive restriction endonuclease digestion of hydroxymethylated DNA. The resulting photocleaved DNA was fully digested by REs, therefore 2-nitrobenzyl...