The Presence of an RNA:DNA Hybrid That Is Prone to Slippage Promotes Termination by T7 RNA Polymerase

•T7 RNA polymerase slips efficiently in polyU and polyA tracts.•Addition of G residue downstream of slipped RNA results in arrested complex.•Slippage-prone tract downstream of stem-loop structure promotes termination.•Clash of stem-loop promotes transcript shearing, misaligned active site, and arrest.•Related yeast mitochondrial RNA polymerase slips poorly, terminates poorly.

Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem–loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem–loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a “locked” complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (85 K)Download as PowerPoint slide