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J. Biol. Chem., Vol. 278, Issue 32, 29710-29718, August 8, 2003

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On the Role of the Escherichia coli RNA Polymerase ⁷⁰ Region 4.2 and -Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter^*

Leonid Minakhin  and Konstantin Severinov   ¶

From the Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854

Bacterial promoters of the extended –10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended –10 Escherichia coli galP1 promoter at temperatures as low as 6 °C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended –10 motif and/or containing the –35 promoter consensus element. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no -subunit C-terminal domains (CTD) and either wild-type ⁷⁰ subunit or ⁷⁰ lacking region 4.2 was assembled and tested for promoter complex formation. At 37 °C, CTD and ⁷⁰ region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended –10 motif. However, no promoter complexes formed when both CTD and ⁷⁰ region 4.2 were absent. Thus, in the context of an extended –10 promoter, CTD and ⁷⁰ region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, CTD and ⁷⁰ region 4.2 interactions with upstream DNA were found to be functionally distinct, for ⁷⁰ region 4.2 but not CTD was required for open promoter complex formation on galP1 derivatives with extended –10 motif. We propose a model involving ⁷⁰ region 4.2 interaction with the flap domain that explains these observations.

Received for publication, May 9, 2003 , and in revised form, June 9, 2003.

^* This work was supported by National Institutes of Health Grant RO1 GM59295 (to K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Waksman Institute, 190 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-6095; Fax: 732-445-5735; E-mail: severik{at}waksman.rutgers.edu.

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