Specificity of release by Escherichia coli transcription termination factor rho of nascent mRNA transcripts initiated at the lambda PR

Abstract

We have studied the specificity and kinetics of release of nascent RNA from ternary transcription complexes by Escherichia coli transcription termination factor rho in vitro. Stable ternary complexes, initiated at the lambda PR promoter, were prepared either by quenching the elongation reaction with EDTA or by preventing further elongation by incorporating 3'-O-methyl nucleotides at the 3' end of the nascent RNA chains. We find that rho protein can only release lambda PR-initiated transcripts from ternary complexes in which transcription has proceeded beyond 288 base pairs from PR; shorter chains are not released. Substitution of inosine for guanosine in the nascent RNA permits the rho-dependent release process to operate on complexes located as close as 108-116 base pairs downstream from PR. The regions of the template from which rho can release transcripts correspond, for both guanosine- and inosine-containing RNA, to those within which rho-dependent termination has also been shown to occur (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553-9564, 9565-9574). The half-time for the major part of the release process is less than 10 s. These results are in good accord with the hypothesis that the specificity of rho-dependent termination is jointly determined by two separable processes: (i) the specificity of rho binding to the nascent RNA chain and (ii) the location and strength of RNA polymerase-pausing sites.