Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters.
The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Rockwell
P
P
Department of Biological Sciences, Hunter College of the City University of New York, New York 10021.
Krakow
J S
JS
D000911:Antibodies, Monoclonal; D012321:DNA-Directed RNA Polymerases / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D012321:DNA-Directed RNA Polymerases