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.