Effects of an anti-.alpha. monoclonal antibody on interaction of Escherichia coli RNA polymerase with lac promoters

Abstract

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)

Genetics of eukaryotic RNA polymerases I, II, and III.

“…For RNAPI and RNAPIII, it has been suggested that the role of one a subunit might be carried out by RPC40 whereas that of the other might be carried out by RPC19, which is also present in the polymerase complex at a stoichiometry of 1 (37). The finding that the two a subunits in E. coli RNAP are not functionally identical (140,141) supports the notion that RPC40 and RPC19, together, form the equivalent of the bacterial a dimer, even though they share only one of the four a-homology regions and that both proteins are structurally different outside of this region.…”

Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium

“…region of the a subunit is available in the assembled RNA polymerase for interaction with other proteins. This same monoclonal antibody interferes with the interaction between the polymerase and the CRP-cAMP complex at the lac promoter, resulting in inhibition of transcription initiation (33). Although other interpretations have not been excluded, it is possible that binding of the antibody interferes with an interaction of the a subunit with CRP.…”

The α subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli

“…The OBS are of two motifs, F-boxes and C-boxes [14] and these boxes have different affinities for OmpR. Riftina et al [17] and Dalla Venezia and Krakow [18] were able to demonstrate inhibition of specific transcripts with monoclonal antibodies to alpha. The ompF promoter contains three contiguous F boxes and a C-box that is separate and downstream of the F-boxes.…”