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

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.