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Study Of Protein-Protein And Protein-Nucleic Acid Interaction In Regulation Of Gene Expression

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Title Study Of Protein-Protein And Protein-Nucleic Acid
Interaction In Regulation Of Gene Expression
 
Creator Roy, Neeladri Sekhar
 
Subject Structural Biology & Bioinformatics
 
Description A complex network of protein-protein and protein-nucleic acid interaction lies at the root of all steps leading to regulation of gene expression such as transcription,
translation etc. Studying the structural basis behind these interactions and how these interactions regulate the gene expression has been the central theme of our work. We
have tried to get insights in two separate but related areas. In the first case we have tried to understand the basis of protein-DNA interaction which is occurring at the
initial step of prokaryotic transcription initiation, that is, the formation of a closed complex between promoter and RNA polymerase. The other area is in a eukaryotic
system where we have used a chemical genetic approach to intervene into certain protein-protein interactions which are crucial components in signaling cascades
leading to cellular events like growth, proliferation or apoptosis and cell death. By introducing some designed peptide mimic we have tried to see whether we can alter
the gene expression level so as to counter the diseased state with abnormal cell proliferation. a) Probing the mode of DNA-Protein interaction in the first step of prokaryotic
transcription initiation Even in prokaryotes, initiation of transcription is a multistep process- In contrast to the open complex, our understanding of the closed complex is relatively limited because of its transient nature. The mode of recognition of the promoter DNA by RNA polymerase holoenzyme during this very first step of formation of the closed complex has been the objective of our work here. Previous reports indicated that the sub-region 2.4 of σ70, a promoter specificity determining subunit of RNA polymerase
holoenzyme, contacts nucleotide sequences in the -10 region of promoter DNA. We have tried to find the differences in the binding constants for the initial closed complex formation for a series of mutants in the -10 region of promoters and sub region 2.4 of σ70 using the quantitative technique of fluorescence anisotropy. This was done to find out whether any specific base-amino acid interaction actually occurs between the RNA polymerase holoenzyme and duplex promoter or is it the overall shape that is recognized during formation of the closed complex.
b) Interrupting protein-protein interactions with synthetic peptide mimics Protein-protein interaction networks play a key role in a large number of cellular processes. The underlying cause of many diseases is found in defective proteinprotein interactions and the resulting deregulated signaling. Inhibition of proteinprotein interactions that rebalance the dysregulated pathway is thus crucial to combat
the disease processes. Here in this work we have tried to intervene into two specific protein-protein interactions viz p53-mdm2 and ras-sos interactions, each of which has
been crucial components of signaling cascades regulating cell proliferation. Since both these interactions are reported to occur involving a helix, one from the p53 Nterminal domain and the other from Sos respectively, we have designed peptide mimics with enhanced helicity by inserting unnatural amino acids like Aib and manipulating some charged residues at non-interacting positions. One more reason behind using amino acids like Aib has been to increase the peptide stability within the cell. By varied manipulations we have found one peptide mimic with potent inhibitory property in each case. Their effects and IC50 value in cancer cell lines like A549 and SKmel5 look promising for further development.
 
Date 2013
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2060/1/FINAL__NEELADRI_SEKHAR_ROY.pdf
Roy, Neeladri Sekhar (2013) Study Of Protein-Protein And Protein-Nucleic Acid Interaction In Regulation Of Gene Expression. PhD thesis, Calcutta University.
 
Relation http://www.eprints.iicb.res.in/2060/