Identification of RNA polymerase β' subunit segment contacting the melted region of the lacUV5 promoter

Abstract

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 β' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of β' and σ70 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 β' subunit were mapped to the N-terminal segment (Cys198-Met237), which is contiguous to the evolutionary conserved region B. The proximity of the β' and σ crosslinks suggest that the N-terminal region of the β' subunit, interacting with single-stranded promoter DNA, can cooperate with the σ subunit in the process of open complex formation.