E.coli RNA polymerase, deleted in the C-terminal part of its α-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the gaP1 promoter

Annie Kolb, Kazuhiko Igarashi, Akira Ishihama, Marc Lavigne, Malcolm Buckle, Henri Buc

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

A deletion of the C-terminal part of the a-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at - 61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at ga/P1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have Investigated the differences In the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated a-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and ga/P1 promoters (respectively lacUV5 and ga/9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated α-subunits, show cooperative binding at the ga/P1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position-40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the ga/P1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the a-subunit is not involved in activation.

Original languageEnglish
Pages (from-to)319-326
Number of pages8
JournalNucleic acids research
Volume21
Issue number2
DOIs
Publication statusPublished - 1993 Jan 25
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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