Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization.
In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.
Cannon
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Department of Biology, Biomedical Sciences Building, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, UK.
Gallegos
M T
MT
Casaz
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P
Buck
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M
D004247:DNA; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D020035:Holoenzymes; D009692:Nucleic Acid Heteroduplexes; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012697:Serine Endopeptidases / Q000378:metabolism; D012808:Sigma Factor / Q000378:metabolism*; D013698:Templates, Genetic; D015533:Transcriptional Activation