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The hydrophobic heptad repeat in Region III of Escherichia coli transcription factor sigma 54 is essential for core RNA polymerase binding

Institute of Medicine¹ and School of Medical Technology², Chung Shan Medical and Dental College, Taichung, Taiwan, ROC

Author for correspondence: Mingli Hsieh. Tel: +886 4 3896190 ext. 50617. Fax: +886 4 3892412. e-mail: mingli{at}mercury.csmc.edu.tw

Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the -12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30–40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the -12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of -12 promoter element recognition and mRNA production.

Keywords: sigma 54, site-specific mutagenesis, hydrophobic heptad repeat, enhancer-dependent transcription, core-binding activity

Abbreviations: DMS, dimethyl sulfate

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