Systematic analysis of σ54 N-terminal sequences identifies regions involved in positive and negative regulation of transcription

The conserved amino-terminal region of sigma 54 (Region I) contains sequences that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to systematically define Region I elements that contribute to each of these functions,,Amino acid residues from 6 to 50 were substituted with alanine in groups of three consecutive residues, making a total of 15 mutants. Mutants were tested for their ability to mediate activation in vivo, and in vitro, and to support transcription in the absence of activator in vitro. Most mutations located between residues 15 and 47 altered sigma function, while mutations between residues 6 and 14, and 48-50 had little effect. The defective mutants ala 15-17, 42-44, and 45-47 define new amino acids required for normal sigma function. In general, there is an inverse correlation between the levels of activated and activator-independent transcription, suggesting that the two functions are linked. When activated, the defective sigma mutants, except for ala 24-26, formed heparin-resistant open complexes similar to wild-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, suggesting that this mutation interferes with a different step in the initiation pathway. (C) 1999 Academic Press.