Probing the informational content of Escherichia coli sigma 70 region 2.3 by combinatorial cassette mutagenesis

Combinatorial cassette mutagenesis was used to probe the informational content of region 2.3 of sigma 70, the RNA polymerase subunit that confers promoter specificity. Region 2.3 is highly conserved among major sigmas of diverse eubacteria, and has been predicted to have a role in melting the DNA duplex around the startpoint of transcription. This prediction was based on sequence similarity with the RNP-1 (ribonucleoprotein) motif of eukaryotic single-stranded RNA-binding proteins, and the abundance of aromatic and basic residues that could potentially interact with the single-stranded DNA. The mutagenesis technique used here consists of simultaneously mutagenizing several codons by cloning synthetic DNA cassettes, and characterizing the rare mutants that retain activity. The results show that most residues in region 2.3 are surprisingly tolerant of amino acid substitutions, including several conserved aromatics and other residues that match the RNP-1 motif. These conserved residues are not essential for transcription even at 17 degrees C, where the DNA melting step is more likely to be rate-limiting. In contrast, Thr429 is quite intolerant to substitution and is predicted to have an important role in sigma 70 function.