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J Biol Chem, Vol. 275, Issue 5, 3661-3666, February 4, 2000

Identification of RNA Polymerase ' Subunit Segment Contacting the Melted Region of the lacUV5 Promoter^*

Konstantin Brodolin^§, Arkady Mustaev^¶, Konstantin Severinov, and Vadim Nikiforov

From the  Institute of Molecular Genetics Russian Academy of Sciences, Kurchatov Sq. 46, Moscow 123182, Russia, the ^¶ Public Health Research Institute, New York, New York 10016, and the  Waksman Institute, State University of New Jersey, Rutgers, Piscataway, New Jersey 08854

Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase ' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of ' and ⁷⁰ subunits at positions 5 and 3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the ' subunit were mapped to the N-terminal segment (Cys¹⁹⁸-Met²³⁷), which is contiguous to the evolutionary conserved region B. The proximity of the ' and  cross-links suggest that the N-terminal region of the ' subunit, interacting with single-stranded promoter DNA, can cooperate with the  subunit in the process of open complex formation.

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