Mutant Forms of Salmonella typhimurium ς(54) Defective in Transcription Initiation but Not Promoter Binding Activity

Transcription initiation with ς(54)-RNA polymerase holoenzyme (ς(54)-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. ς(54)’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 ς(54) that were deficient in transcription initiation but still directed ς(54)-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 ς(54)-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of ς(54) 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.