Sequences within the DNA cross-linking patch of σ54 involved in promoter recognition, σ isomerization, and open complex formation

Abstract

The bacterial RNA polymerase holoenzyme containing the σ54 subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the σ54 DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the σ and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by σ54, and holoenzyme and is involved in activator-dependent σ isomerization. As part of the σ54-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the -12 promoter region to set basal and activated levels of transcription.