Initiation of transcription is a major step in the regulation of gene expression. A dominant theme in regulation of gene expression lies in understanding the mechanism involved in selective expression of the genes in response to external or internal stimuli. Gene regulatory proteins bind DNA at specific sites either cognate to the promoters they act upon or at a distance, thereby exerting their effect by turning on (activation) or turning off (repression) the genes. Response of these factors to the environmental signals is further achieved by the DNA binding affinity of the transcription factors that can be modulated by small ligands, concentrations of which may fluctuate in response to nutrient availability and stress. Bacteriophages achieve a high degree of efficiency in gene expression by evolving elegant strategies of transcriptional control. mom gene of enterobacteriophage Mu serves as an excellent model to understand this elaborate regulation of gene expression. The gene encodes a unique DNA modification function that confers an anti-restriction phenotype to the phage genome. Though dispensable for phage growth, it is fascinating in two respects (i) a novel modification; (ii) regulation follows a complex scheme without precedence in prokaryotes. mom is the last gene to be expressed during the phage lytic life cycle. Premature expression of the gene is deleterious to both host and phage and hence it is under a complex regulatory network. Dam methylase, a host encoded protein acts as a positive regulator of gene expression, an example where methylation has been shown to play a positive role in regulating tranascription. OxyR, another host encoded protein negatively regulates mom gene expression. Dam methylation prevents the binding OxyR to its site located in the mom regulatory region. The regulatory interplay also involves two phage encoded proteins. C, a middle gene product is essential for transcriptional switch from middle to late genes and Com, a late gene product, for enhancing translation of mom mRNA. Thus, C and Com serve as transcriptional and translational activators of mom gene expression. Pmom is a weak promoter with both -10 and -35 elements away from consensus and a sub-optimal 19 bp spacer element encompassing a stretch of 6T residues that act as negative elements. ‘T stretch’ is known to induce a kink in the DNA. The sub-optimal spacer region makes the promoter elements out of phase and RNAP by itself cannot bind at mom promoter. C protein exerts its effect in activation in a multistep mechanism. The protein binds DNA as a dimer overlapping the promoter and unwinds the DNA, realigning the promoter elements, thus recruiting the RNAP. In the next step, it enhances the promoter clearance by the enzyme, thus enhancing the rate of productive transcription. With this prevailing knowledge on C mediated mom gene expression, the present thesis work describes the experiments carried out to further understand the molecular mechanism of second step activation at Pmom. Genetic and biochemical analysis were carried out to identify the interacting surface of C protein on RNAP. Subsequently, studies have been extended to understand the C mediated transactivation at other late promoters- lys, I, P, which encode for the lysis and morphogenetic functions of the phage. Finally, Mg2+ coordinating residues in C protein were identified to decipher the ligand induced conformational changes in the activator protein required for its transactivator function. Chapter I, a general introduction to the thesis, deals with the detailed discussion on gene expression and its regulatory mechanisms. RNA polymerase (RNAP) being the central molecule of gene expression (transcription) its organization and assembly are discussed. With the availability of the high resolution crystal structures of bacterial RNAP, an in-depth review on RNAP structure in terms of its potential regulatory targets, conformational changes associated with the formation of a functional holoenzyme, and during its transition from initiation to elongation processes have been described. Regulation of transcription with an emphasis on activation mechanism, ligand mediated allosteric transitions in regulatory proteins and the polymerase-activator interactions are discussed citing a few examples. The chapter concludes by introducing bacteriophage Mu and mom gene and its regulation by C. The objectives of the thesis form the concluding section of the chapter. Activators are capable of resurrecting defective promoters in response to cellular demands. The unusual, multistep activation of mom promoter (Pmom) by C protein involves activator mediated promoter unwinding to recruit RNA Polymerase (RNAP) and subsequent enhanced promoter clearance of the enzyme. The first step of transactivation is an interaction independent step, while the later might involve a transient interaction between C and one of the subunits of RNAP. Previous studies pointed out β′ subunit to be the most probable interaction partner. Chapter II comprises the genetic and biochemical studies carried out to confirm this observation. Employing a genetic screen mutations in rpoC gene (encoding the β′ subunit of RNAP), were isolated which result in the defective RNAP. The mutant RNAPs were assayed for their C specific activity by in vivo transactivation assays. Such mutants have been purified and characterized to understand their effect at different steps of C mediated mom gene expression during transcription initiation. The mutant RNAP had normal transcription activity with typical σ70 promoters but exhibited reduced productive transcription and enhanced abortive initiation on C-dependent Pmom. Experiments carried out to probe the interaction between C and mutant RNAP revealed that the physical interaction per se is not disrupted between the two proteins. Post C-mediated recruitment of RNAP to the promoter, transient interactions between the two proteins appears to induce subtle conformational changes in RNAP leading to an enhanced promoter clearance. Transactiavtor protein C is essential for the expression of other late genes lys, I, P apart from mom during the phage life cycle. Although the mechanism of multistep activation at Pmom has been elucidated, little is known on the transactivation from lys, I and P promoters. Chapter III includes studies carried out to understand the process of activation at these promoters. Owing to the differences in their C-binding site and promoter architecture it was important to investigate the differential effect of C, if any at lys, I , P promoters compared to that at Pmom. Activators in prokaryotes are shown to stimulate different steps of transcription initiation pathway ranging from the polymerase binding to the promoters to the post recruitment steps of isomerization and promoter clearance. Effect of C at different steps of transcription initiation pathway was analysed. The results indicate that C is absolutely essential for transcription from lys, I and P promoters similar to mom. However, at these promoters C exerts its effect at the step of Isomerisation from closed complex to open complex formation. Thus, C acts at a single step here and the mode of activation is different from that observed at Pmom. C dimer binds DNA with high affinity and sequence specificity, to an interrupted palindromic sequence overlapping the -35 element of mom promoter. Mg2+ mediated conformational transitions in C protein are essential for its DNA binding and transactivation functions. Chapter IV deals with the identification of the Mg2+ coordinating residues in C protein. Primary sequence analyses lead to the identification of a putative metal coordinating motif (EXDXD) towards the N-terminus of the protein. These residues were subjected to site directed mutagenesis to infer their role in Mg2+ coordination, its associated allosteric transition required for specific interaction with DNA. Mutants showed an altered Mg2+ induced conformation, compromised DNA binding and reduced levels of transcription activation when compared to C protein. Though Mg2+ is widely used in various DNA transaction reactions, this study provides the first insights on the importance of metal-ion induced allosteric transitions in regulating transcription factor function.

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.