Alteration in template recognition by Escherichia coli RNA polymerase lacking the ω subunit: a mechanistic analysis through gel retardation and foot-printing studies

Mukherjee, Kakoli ; Chatterji, Dipankar (1999) Alteration in template recognition by Escherichia coli RNA polymerase lacking the ω subunit: a mechanistic analysis through gel retardation and foot-printing studies Journal of Biosciences, 24 (5). pp. 453-459. ISSN 0250-5991

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Official URL: http://www.ias.ac.in/jbiosci/december1999/article9...

Related URL: http://dx.doi.org/10.1007/BF02942656

Abstract

The ω subunit ofEscherichia coli RNA polymerase is a 91 amino acid polypeptide which co-purifies with the enzyme and is thought to help in maturing the rest of the enzyme to its full functionality. Purified ω when added externally was found to inhibit general transcriptional activity of ω-less RNA polymerase as well as promoter-specific single-round transcriptional activity at all the promoters tested. In this study we have tried to analyse the observed inhibition of transcription using gel retardation assays and KMnO4foot-printing. Further, through protein foot-printing we have attempted to identify alterations in the interaction of the ω-less core enzyme with the σ70 subunit. Our results suggest that the ω-less holoenzyme has lesser affinity towards the DNA template and external addition of ω destabilizes the open complex for both the wild-type and ω-less enzyme. The ω -less core enzyme interacts with the σ70 subunit to expose the - 35 recognition domain (domain 4.2) unlike that observed in the wild-type interaction. Thus the absence of the ω subunit leads to the formation of an enzyme which has altered DNA binding and σ70 binding properties. Circular dichroic measurements also indicate a major conformational alteration of both holo and core RNA polymerase in the presence and absence of the ω subunit.

Item Type:Article
Source:Copyright of this article belongs to Indian Academy of Sciences.
Keywords:ω Subunit; ω-less RNA Polymerase; Interaction; Promoter; Foot-printing
ID Code:6445
Deposited On:20 Oct 2010 10:23
Last Modified:08 Feb 2011 09:03

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