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

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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 r(A-U)n. DNase I footprinting shows that the preformed mAb 126C-6-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. After incubation of the preformed mAb 126C6-RNA polymerase with cAMP-CRP-lac P+ the cytosines near the transcription start site remain inaccessible to methylation. After incubation of the preformed cAMP-CRP-lac P+-RNA polymerase open complex with mAb 126C6 the cytosines near the start site remain unpaired.

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