Systematic analysis of sigma(54) N-terminal sequences identifies regions involved in positive and negative regulation of transcription

The conserved amino-terminal region of sigma 54 (Region I) contains sequenc es that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to system atically define Region I elements that contribute to each of these function s,,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 t o support transcription in the absence of activator in vitro. Most mutation s located between residues 15 and 47 altered sigma function, while mutation s between residues 6 and 14, and 48-50 had little effect. The defective mut ants 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 le vels 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 wi ld-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, sug gesting that this mutation interferes with a different step in the initiati on pathway. (C) 1999 Academic Press.