A Study OF Mechanism of Transcription

Debnath, Subrata (2011) A Study OF Mechanism of Transcription. PhD thesis, Calcutta University.


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    Roy, Siddhartha


    The transcription is the process by which all forms of cellular RNA are synthesized by RNA polymerases that take instruction from DNA templates. The regulation of transcription depends upon specific protein-nucleic acid and protein-protein interactions. So elucidation of the structural basis of these interactions is of prime importance. In this thesis we have investigated these interactions using two different model systems- the bacterial promoter-RNA polymerase interaction and the human PC4-p53 interaction. The transcription is mainly divided into three steps, initiation, elongation and termination. In the initiation step, RNA polymerase binds to the DNA molecule at a particular site called promoters, which are specific sequences of about 40 base-pairs. The most crucial interactions for positioning of E.coli RNA polymerase at a promoter occur at two shortsequence patches on the DNA. In case of -10/-35 class promoters-------- the -10 hexamer and the -35 hexamer, which are located 10 and 35 base pairs (bp) upstream from the transcription start site, respectively, are important for recognition by RNA polymerase. Extended -10 promoters are somewhat different in architecture. Extended -10 class promoters contain a single -10 consensus element and the -35 region is not critical for promoter activity. In this thesis we have studied the interactions of E.coli RNA polymerase with a -10/-35 class promoter, lambda PR and an extended -10 class promoter, galP1, to elucidate the base positions that are important for RNA polymerasen recognition of -10/-35 class promoter and extended -10 class promoter. We have shown that the initial recognition of the promoter by RNA polymerase is very weakly basespecific and the base positions which are important for recognition by RNA polymerase to galP1 promoter at the initial stage are not similar in case of lambda PR promoter i.e. ecognition pattern of RNA polymerase to galP1 and lambda PR promoter are different. The p53 protein acts as a transcriptional activator of genes containing p53 binding sites and activates transcription of a number of genes which encode for proteins involved in cell cycle arrest, apoptosis and DNA repair in response to DNA damage and other genotoxic insult. The multifunctional human transcriptional coactivator PC4 interacts with p53 and activates several p53-dependent genes. PC4 interacts mainly with the Cterminal negative regulatory domain of p53 through its DNA binding C-terminal half. In this thesis we have investigated which residues in the p53 (364-393) primarily involved in the interaction with PC4 and vice versa. We have shown that amino acids 380-386 of p53 are crucial for interaction with PC4 and serine 73 of PC4 is an important residue for recognition of p53. Intermolecular Nuclear Overhauser Effect placed aspartate 76 of PC4 in the vicinity of lysine 381 of p53, indicating that the region around residues 73–76 of PC4 is important for p53 recognition. We have also studied the binding pattern of PC4 with differently acetylated p53 (380-386) peptides and shown that acetylation of Lys-381 and Lys-382 enhanced the binding by about an order of magnitude.

    Item Type: Thesis (PhD)
    URI: http://www.eprints.iicb.res.in/id/eprint/1601
    Subjects: Structural Biology & Bioinformatics
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Mr Santanu Sadhukhan
    Date Deposited: 30 Aug 2012 15:10
    Last Modified: 30 Aug 2012 15:10

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