Mutant forms of Salmonella typhimurium σ54 defective in transcription initiation but not promoter binding activity

Abstract

Transcription initiation with σ54-RNA polymerase holoenzyme (σ54- holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. σ54's binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium σ54 that were deficient in transcription initiation but still directed σ54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for σ54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of σ54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.