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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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|
|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|
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