An insertion in the catalytic trigger loop gates the secondary channel of RNA polymerase

Ran Furman, Oleg V. Tsodikov, Yuri I. Wolf, Irina Artsimovitch

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Escherichia coli DksA and GreB bind to RNA polymerase (RNAP), reaching inside the secondary channel, with similar affinities but have different cellular functions. DksA destabilizes promoter complexes whereas GreB facilitates RNA cleavage in arrested elongation complexes (ECs). Although the less abundant GreB may not interfere with DksA regulation during initiation, reports that DksA acts during elongation and termination suggest that it may exclude GreB from arrested complexes, potentially triggering genome instability. Here, we show that GreB does not compete with DksA during termination whereas DksA, even when present in several hundredfold molar excess, does not inhibit GreB-mediated cleavage of the nascent RNA. Our findings that DksA does not bind to backtracked or active ECs provide an explanation for the lack of DksA activity on most ECs that we reported previously, raising a question of what makes a transcription complex susceptible to DksA. Structural modeling suggests that i6, an insertion in the catalytic trigger loop, hinders DksA access into the channel, restricting DksA action to a subset of transcription complexes. In support of this hypothesis, we demonstrate that deletion of i6 permits DksA binding to ECs and that the distribution of DksA and i6 in bacterial genomes is strongly concordant. We hypothesize that DksA binds to transcription complexes in which i6 becomes mobile, for example, as a consequence of weakened RNAP interactions with the downstream duplex DNA.

Original languageEnglish
Pages (from-to)82-93
Number of pages12
JournalJournal of Molecular Biology
Volume425
Issue number1
DOIs
StatePublished - Jan 9 2013

Bibliographical note

Funding Information:
We thank Seth Darst for providing a model of E. coli GreB bound to EC and Georgy Belogurov and the members of the Gourse laboratory for stimulating discussions. This work was supported by the National Science Foundation ( MCB-0949569 ; I.A.) and by the Intramural Research Program of the National Library of Medicine at National Institutes of Health (Y.I.W.).

Keywords

  • DksA
  • Gre
  • RNA polymerase
  • secondary channel
  • trigger loop

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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