Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein.
The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.
Liu
Mofang
M
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264, USA. liumo@pop.nci.nih.gov
Gupte
Geeta
G
Roy
Siddhartha
S
Bandwar
Rajiv P
RP
Patel
Smita S
SS
Garges
Susan
S
D015075:2-Aminopurine / Q000737:chemistry; D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D015075:2-Aminopurine; D012321:DNA-Directed RNA Polymerases