Pathway of promoter melting by Bacillus subtilis RNA polymerase at a stable RNA promoter: effects of temperature, delta protein, and sigma factor mutations.
Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (beta beta' alpha 2; or E) associated with one of several sigma factors, which determine promoter recognition, and delta protein, which enhances promoter selectivity. We have shown previously that specific mutations in sigma A region 2.3, or addition of delta, decrease the ability of RNAP to melt the ilv-leu promoter. Here we extend these studies to a stable RNA promoter, PtmS, which controls transcription of seven tRNA genes. KMnO4 footprinting was used to visualize DNA melting at PtmS as a function of both temperature and the protein composition of the RNAP holoenzyme. We propose that the pathway leading to productive initiation includes several intermediates: a closed complex (RPc), a complex in which DNA melting has nucleated within the conserved TATA element (RPn), and an open complex in which DNA-melting extends to at least -4 (RPo1). RNAP reconstituted with either of two mutant sigma A proteins, Y189A and W192A, was defective for both the nucleation and propagation of the transcription bubble while a third sigma A mutant, W193A, allows normal nucleation of DNA-melting, but does not efficiently propagate the melted region downstream.
Juang
Y L
YL
Section of Microbiology, Cornell University, Ithaca, New York 14853-8101, USA.
Helmann
J D
JD
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009876:Operon; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012808:Sigma Factor / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016385:TATA Box; D013816:Thermodynamics; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I