Effects of an anti-alpha monoclonal antibody on interaction of Escherichia coli RNA polymerase with lac promoters.
The anti-alpha monoclonal antibody, mAb 126C6, has been used to investigate the role of the alpha subunit in transcription initiation. mAb 126C6 strongly inhibits cAMP-CRP-dependent abortive initiation with lac P+, partially inhibits abortive initiation with the lac L8UV5 promoter, and is without effect on the d(A-T)n-directed synthesis of r(A-U)n. DNase I footprinting shows that the preformed mAb 126C6-RNA polymerase complex does not bind to cAMP-CRP-lac P+; RNA polymerase specific protection is largely lost after incubation of the preformed RPo with mAb 126C6. Kinetic analysis of open complex formation by mAb 126C6-RNA polymerase with lac L8UV5 showed that changes in both the binding and the rate of isomerization account for the observed inhibition, with the isomerization step affected to a greater extent. Binding of cAMP-CRP to lac L8UV5 is RNA polymerase dependent. DNase I footprints show that as a consequence of mAb 126C6 binding of the preformed cAMP-CRP-lac L8UV5-RNA polymerase RPo, CRP dissociates from its site on the promoter. RNA polymerase protection of the promoter upstream from -41 is also lost. DNase I footprinting of mAb 126C6-RNA polymerase complexed with cAMP-CRP-lac P+ or -lac L8UV5 suggests that interactions between CRP and RNA polymerase are affected by binding of the anti-alpha mAb 126C6 to RNA polymerase. Protection methylation studies demonstrate that the formation of the mAb 126C6-RNA polymerase-lac L8UV5 open complex occurs at a slower rate and that nonoptimal contacts are established between mAb 126C6-RNA polymerase-lac L8UV5 promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Riftina
F
F
Department of Biological Sciences, Hunter College of the City University of New York, New York 10021.
DeFalco
E
E
Krakow
J S
JS
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D012321:DNA-Directed RNA Polymerases