Functionally distinct RNA polymerise binding sites in the phage Mu mom promoter region

Balke, Virginia and Nagaraja, Valakunja and Gindlesperger, Tracy and Hattman, Stanley (1992) Functionally distinct RNA polymerise binding sites in the phage Mu mom promoter region. In: Nucleic Acids Research, 20 (11). pp. 2777-2784.

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Abstract

Transcription of the phage Mu com/mom operon is trans-activated by another phage gene product, C, a site-specific DNA binding protein. To gain insight into the mechanism by which C activates transcription, we carried out footprinting analyses of Escherichia coil RNA polymerase (= RNAP) binding to various com-lacZ fusion plasmids. KMnO4-sensitive sites (diagnostic of the melted regions in open-complexes) and DNase (- sensitive sites were located by primer-extension analysis. The results are summarized as follows: (i) In vivo, in the absence of C, RNAP bound in the wild-type (wt) promoter region at a site designated P2; in vitro DNase I-footprinting showed that P2 extends from – 74 to - 24 with respect to transcription initiation. This overlaps a known strong C-binding site (at -35 to - 54). RNAP bound at P2 appeared to be in an opencomplex, as evidenced by the presence of KMnO4-hypersensitive sites. (ii) In contrast, when C was present in vivo, RNAP bound in the wt promoter region at a different site, designated P1, located downstream and partially overlapping P2. RNAP bound at P1 also appeared to be in an open-complex, as evidenced by the presence of KMnO4-hypersensitive sites. (iii) Two C-independent mutants, which initiate transcription at the same position as the wt, were also analyzed. In vivo, in the absence of C, RNAP bound mutant tin7 (contains a T to G substitution at -14) predominantly at P1; in vitro DNase I-footprinting showed that P1 extends from -56 to + 21. With mutant tin6 (a 63 base-pair deletion removing P2, as well as part of P1 and the C-binding site from - 35 to - 54), RNAP bound to P1 independent of C. We conclude that P1 is the `functional' RNAP binding site for momtranscription initiation, and that C activates transcription by promoting binding at P1, while blocking binding at P2.

Item Type:	Journal Article
Publication:	Nucleic Acids Research
Publisher:	Oxford University Press
Additional Information:	Copyright for this article belongs to Oxford University Press.
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	10 Dec 2004
Last Modified:	19 Sep 2010 04:16
URI:	http://eprints.iisc.ac.in/id/eprint/1979

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