Amino-terminal sequences of sigma(N) (sigma(54)) inhibit RNA polymerase isomerization

In bacteria, association of the specialized sigma(N) protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but u nable to melt DNA and initiate transcription unless acted on by an activato r protein. The conserved amino-terminal 50 amino acids of sigma(N) (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 tra nscription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I s equences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigma(N)-holoenzyme o r removal of Region I allowed efficient formation of complexes in which mel ted DNA was sequestered by RNA polymerase. Like natural pre-initiation comp lexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The in hibitory sigma(N) Region I domain functioned in trans to confer heparin sen sitivity to complexes between Region I-deleted holoenzyme and pre-melted pr omoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one functi on for Region I is to mask a single-strand DNA-binding activity of the holo enzyme. On the basis of extended DNA footprints of Region I-deleted holoenz yme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable as sociation of holoenzyme with melted DNA.