Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex.
Characterization of ternary complexes containing an Escherichia coli lac promoter DNA fragment, CAP protein and RNA polymerase, separated on non-denaturing polyacrylamide gels and footprinted in the gel slice, reveals a striking stabilization of CAP against dissociation in the open complex, compared to the CAP-DNA complex lacking RNA polymerase. The stabilization is lost when half a helical turn of DNA is inserted between CAP and polymerase sites, but is partially restored with an 11 base-pair insert; stimulation of transcription parallels the stabilization effect. This behavior suggests a direct protein-protein interaction. Comparison of initiation kinetics for wild-type and a mutant in which the P2 promoter has been inactivated shows that CAP both strengthens binding in the closed complex and accelerates isomerization to the open complex; the latter effect accounts for the bulk of the observed transcriptional activation.
Straney
D C
DC
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Straney
S B
SB
Crothers
D M
DM
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004357:Drug Synergism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D007763:Lac Operon; D009154:Mutation; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011506:Proteins; D011956:Receptors, Cell Surface; D014157:Transcription Factors; C481662:crp protein, E coli; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I