Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase.
Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(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; D000915:Antibody Affinity; D001667:Binding, Competitive; D012321:DNA-Directed RNA Polymerases / Q000276:immunology*; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D012321:DNA-Directed RNA Polymerases