CASSETTE MUTAGENESIS IMPLICATES A HELIX-TURN-HELIX MOTIF IN PROMOTER RECOGNITION BY THE NOVEL RNA-POLYMERASE SIGMA-FACTOR SIGMA-54

Cassette mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma-54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma-54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo foot-printing indicated that the inactive HTH mutants of sigma-54 were defective interaction with both the -24 and -12 regions of the glnAp2 promoter.