Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides.
The specific recognition by Escherichia coli RNA polymerase of single-stranded oligodeoxyribonucleotides (oligos) with the sequence of the -10 promoter region on the nontemplate strand has been studied. Binding was monitored by observing the increase in fluorescence of 2-aminopurine residues incorporated in the oligos. The effects of salt on the rates of formation and dissociation of RNA polymerase.oligo complexes are relatively small, from which we conclude that electrostatic interactions contribute minimally to the favorable binding free energy. From the convex temperature dependence of ln Ka (Ka is the equilibrium association constant), we infer that a large apparent negative heat capacity, of 1-2 kcal M-1 K-1, accompanies complex formation, which is interpreted as due to a conformational change in RNA polymerase. Contrary to expectation, the forward rate constant for binding of oligos is more than 10-fold smaller than that for open complex formation at strong promoters. This suggests that in comparison to an oligo, promoter DNA may be better able to accelerate this required conformational change in the RNA polymerase. Oligo binding was shown to compete with the interaction between RNA polymerase and promoters, indicating that the two bind to overlapping sites on the RNA polymerase
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
A M
AM
Fedoriw
H
H
Liu
V E
VE
Anderson
P L
PL
deHaseth
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013698:Templates, Genetic; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D012321:DNA-Directed RNA Polymerases