The ability to fine-tune gene-expression in the adverse conditions during pre and post infectious stages has contributed in no small measure to the success of Mycobacterium tuberculosis as the deadly pathogen. Multiple sigma factors, transcription regulators, and diverse two component systemshave facilitated tailoring the metabolic pathways to meet the challenges faced by the pathogen. Over the last decade, studies have been initiated to understand the various facets of transcription in mycobacteria. Although not as extensive as the work in other model systems, such as Escherichia coli and eukaryotes, it is evident from these initial studies that the machinery is conserved,yetmany aspects of transcription and its regulation seem to be different in mycobacteria.The work presented in the thesis deals with some of the steps in the process, primarily initiation in the context of the distinct physiology of M. tuberculosis. The detailed kinetic and equilibrium study of a few selected promoters of M. tuberculosis viz.PgyrB1, PgyrR, PrrnPCL1 and PmetU is described in Chapter 2.Different stages of transcription initiation that have been analyzed include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance.The equilibrium binding and kinetic studies of various steps reveal distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. In addition, a novel aspect of the transcription initiation at the gyr promoter was unraveled. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. In Chapter 3, the mechanism of growth phase dependent control (GPDC) at a few of the M. tuberculosis promoters has been investigated. The experiments described in the chapter are carried out to demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of M. tuberculosis to facilitate the iNTPs and pppGpp mediated regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. In chapter 4, the long standing hypothesis that deals with interdependence of the transcription elongation kinetics and the growth rates has been addressed. Previous studies suggest that the rate of synthesis of the key molecules in cells affects the growth kinetics. In order to validate, the kinetics of elongation of RNAPs from M. tuberculosis, M. smegmatis and E. coli whose growth rates vary from very slow to fast is measured. Surface Plasmon Resonance (SPR) is used to monitor the transcription in real time and kinetic equations are applied to calculate the elongation rates. Further, the effects of the composition of the template DNA on the elongation rates of RNAP from E. coli and M. smegmatis, whose genomes show difference in the GC content are explored. The results obtained from the analysis support the hypothesis and also reveal the effect of template composition on elongation rates of RNAP.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/3187 |
| Date | January 2014 |
| Creators | Tare, Priyanka |
| Contributors | Nagaraja, Valakunja |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G26386 |
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