Mapping the molecular interface between the σ70 subunit of E. coli RNA polymerase and T4 AsiA

Leonid Minakhin, Julio A. Camarero, Mande Holford, Christian Parker, Tom W. Muir, Konstantin Severinov

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Bacteriophage T4 antisigma protein AsiA (10 kDa) orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the σ70 subunit of E. coli RNA polymerase. The molecular determinants of σ70-AsiA complex formation are not known. Here, we used combinatorial peptide chemistry, protein-protein crosslinking, and mutational analysis to study the interaction between AsiA and its target, the 33 amino acid residues-long σ70 peptide containing conserved region 4.2. Many region 4.2 amino acid residues contact AsiA, which likely completely occludes the DNA-binding surface of region 4.2. Though none of region 4.2 amino acid residues is singularly responsible for the very tight interaction with AsiA, σ70 Lys593 and Arg596 which lie outside the putative DNA recognition element of region 4.2, contribute the most. In AsiA, the first 20 amino acid residues are both necessary and sufficient for interactions with σ70. Our results clarify details of σ70-AsiA interaction and open the way for engineering AsiA derivatives with altered specificities.

Original languageEnglish (US)
Pages (from-to)631-642
Number of pages12
JournalJournal of Molecular Biology
Volume306
Issue number4
DOIs
StatePublished - Mar 2 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • Antisigma AsiA
  • Bacteriophage T4
  • Crosslinking
  • Polypeptide libraries
  • Transcription regulation

Fingerprint

Dive into the research topics of 'Mapping the molecular interface between the σ<sup>70</sup> subunit of E. coli RNA polymerase and T4 AsiA'. Together they form a unique fingerprint.

Cite this