Single amino acid substitution mutants of Klebsiella pneumoniae sigma(54) defective in transcription

Transcription initiation by the sigma (54) RNA Polymerase requires speciali sed activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of sigma (54) in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered sigma (54) proteins. Five defective mutants, each with a different single amino a cid substitution, were obtained. Three failed in transcription after formin g a closed complex. One such mutant mapped to regulatory Region I of sigma (54), the other two to Region III. The Region I mutant allowed transcriptio n independently of activator and showed reduced activator-dependent sigma ( 54) isomerisation. The two Region III mutants displayed altered behaviour i n a sigma (54) isomerisation assay and one failed to stably bind early melt ed DNA as the holoenzyme; they may contribute to a communication pathway li nking changes in cr to open complex formation, Two further Region III mutan ts showed gross defects in overall DNA binding. For one, sufficient residua l DNA binding activity remained to allow us to demonstrate that other activ ities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.