Monoclonal antibodies as probes of the topological arrangement of the .alpha.-subunits of Escherichia coli RNA polymerase

Abstract

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 residues is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the .omega. 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. These studies suggest that the in vitro assembly of the polymerase core may proceed via the formation of the more stable .alpha..beta. complex followed by addition of the second .alpha. subunit.