Kinetics of the stages of transcription initiation at the Escherichia coli lac UV5 promoter.
The kinetics of initiation by Escherichia coli RNA polymerase on the lac L8UV5 promoter was studied by a gel retardation method that separates protein-DNA complexes from free DNA. The binding constant of the closed complex, the forward and reverse rate constants of isomerization from closed to open complex, and the forward rate constant from the open to initiated complex were measured. Both the forward and reverse isomerization rates were found to be temperature dependent, and the activation energies for these steps were determined. The rates of open complex formation and dissociation were not affected by the addition of ribonucleotide triphosphates; however, the extent of dissociation was greatly reduced if the triphosphates added allowed a short, unstable RNA product to form. The dissociation rate was not affected by heparin, a polyanion competitor that sequesters the polymerase. The rate of initiated complex formation appeared to be dependent on whether the initiating moiety was a mononucleotide triphosphate or dinucleoside monophosphate and on the sequence of the dinucleoside. These results are compared to those found on both the lac L8UV5 and other bacterial and phage promoters by less direct measurements. We use the values obtained for the individual rate constants to investigate the predicted steady-state kinetics of initiation-limited transcription, with the conclusion that the rate-limiting step is formation of the open complex in the limit of low polymerase concentration. However, when RNA polymerase is saturating, the rate is determined by the transition from open complex into the stably initiated ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Straney
S B
SB
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Crothers
D M
DM
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008433:Mathematics; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D012321:DNA-Directed RNA Polymerases