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)

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)