Scanning Mutagenesis Reveals Roles for Helix N of the Bacteriophage T7 RNA Polymerase Thumb Subdomain in Transcription Complex Stability, Pausing, and Termination

Luis G. Brieba, Vijaya Gopal, Rui Sousa

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Deletions within the thumb subdomain (residues 335-408) of T7 RNA polymerase decrease elongation complex stability and processivity, but the structure of a T7RNAP initial transcription complex containing a 3-nucleotide RNA reveals no interactions between the thumb and the RNA or DNA. Modeling of a longer RNA in this structure, using a T7DNAP-primer-template structure as a guide, suggests that the phosphate ribose backbone of the RNA contacts a stretch of mostly positively charged side chains between residues 385 and 395 of helix N of the thumb. Scanning mutagenesis of this region reveals that alanine substitutions of Arg391, Ser393, and Arg 394 destabilize elongation complexes and that substitutions at 393 and 394 increase termination of transcripts 5 or more bases in length. The α-carbons of all 3 of these residues lie on the side of helix N, which faces into the template-binding cleft of the RNA polymerase, and modeling suggests that they can contact the RNA 4-5 bases away from the 3′-end. Alanine substitutions of other residues within 385-395 do not have marked effects on transcription complex stability, but alanine substitutions of Asp388 and Tyr385 reduce pausing and termination at the T7 concatemer junction. Both of these side chains lie on the outer side of helix N, pointing away from the template binding cleft. The thumb subdomain of T7RNAP therefore has roles both in transcription complex stabilization and in pausing and termination at the T7 concatemer junction.

Original languageEnglish (US)
Pages (from-to)10306-10313
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number13
DOIs
StatePublished - Mar 30 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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