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Nucleic Acids Research, 2000, Vol. 28, No. 22 4419-4427
© 2000 Oxford University Press

Single amino acid substitution mutants of Klebsiella pneumoniae {sigma}54 defective in transcription

Melinda Pitt, María-Trinidad Gallegos and Martin Buck*

Department of Biology, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK

Transcription initiation by the {sigma}54 RNA polymerase requires specialised 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 acid substitution, were obtained. Three failed in transcription after forming 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 transcription independently of activator and showed reduced activator-dependent {sigma}54 isomerisation. The two Region III mutants displayed altered behaviour in a {sigma}54 isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in {sigma} to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.

* To whom correspondence should be addressed. Tel: +44 20 7594 5442; Fax: +44 20 7594 5419; Email: m.buck{at}ic.ac.uk Present address: María-Trinidad Gallegos, Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín (CSIC), Profesor Albareda 1, 18008 Granada, Spain


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