MUTATIONAL ANALYSIS OF THE ROLE OF THE FIRST HELIX OF REGION-4.2 OF THE SIGMA(70) SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE IN TRANSCRIPTIONAL ACTIVATION BY ACTIVATOR PROTEIN PHOB

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70 which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif of region 4.2 of sigma(70). To study further the role of the first helix of the HTH motif of sigma(70) in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma(70) proteins. RNA polymerases containing the mutant sigma(70)s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma(70) (E574 K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma(70) mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.