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A structural insight into the prokaryotic heat shock transcription regulatory protein ?(32): an implication of ?(32)-DnaK interaction.

ABSTRACT: The heat shock response mechanism is a very vital biochemical process and is mainly controlled by ?(32) protein. The function of ?(32) is temperature dependent and at lower temperatures ?(32) is inactivated by its interactions with DnaK. This interaction is completely abolished above 42°C till date no molecular details of the interactions are available. In the present scenario, an attempt has been made to analyze first the predicted structure of ?(32) obtained by comparative modeling techniques and then to study the interactions between ?(32) and DnaK. From this molecular modeling study we could specifically identify the binding sites of the interactions of ?(32) with DnaK which will enlighten the mechanism of regulation of its activity and stability by DnaK. Our study provides the idea for future mutational experiments in order to find out the possible roles of the amino acids of region2 and region3 of ?(32) in stability as well as in binding with DnaK.

SUBMITTER: Roy SS

PROVIDER: S-EPMC3524887 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A structural insight into the prokaryotic heat shock transcription regulatory protein σ(32): an implication of σ(32)-DnaK interaction.

Roy Sourav Singha SS Patra Monobesh M Dasgupta Rakhi R Bagchi Angshuman A

Bioinformation 20121031 21

The heat shock response mechanism is a very vital biochemical process and is mainly controlled by σ(32) protein. The function of σ(32) is temperature dependent and at lower temperatures σ(32) is inactivated by its interactions with DnaK. This interaction is completely abolished above 42°C till date no molecular details of the interactions are available. In the present scenario, an attempt has been made to analyze first the predicted structure of σ(32) obtained by comparative modeling techniques ...[more]

PMID: 23275701

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Project description:BACKGROUND:The effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5'-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines. RESULTS:After the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p <?0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5'-flanking region sequence (2633?bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5'-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39?°C and 42?°C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter. CONCLUSIONS:These findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.

Dataset Information

A structural insight into the prokaryotic heat shock transcription regulatory protein ?(32): an implication of ?(32)-DnaK interaction.

Publications

A structural insight into the prokaryotic heat shock transcription regulatory protein σ(32): an implication of σ(32)-DnaK interaction.

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