title: The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.
abstract: 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.
authors: Graduate Program in Cell and Molecular Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA; Department of Cell Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA.
authors: Vadim
authors: V
authors: Molodtsov
authors: Department of Cell Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA.
authors: Michael
authors: M
authors: Anikin
authors: Department of Cell Biology, Rowan University School of Osteopathic Medicine, 42 East Laurel Road, UDP 2200, Stratford, NJ 08084, USA. Electronic address: wtmcallister@gmail.com.
authors: William T
authors: WT
authors: McAllister
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D055029:Inverted Repeat Sequences / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D008957:Models, Genetic; D011061:Poly A / Q000235:genetics; D011072:Poly U / Q000235:genetics; D012313:RNA / Q000235:genetics; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic / Q000235:genetics*; D061806:Transcription Termination, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology
chemical_list: D014764:Viral Proteins; D011061:Poly A; D011072:Poly U; D012313:RNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: RNA pullout; RNA:DNA hybrid; hybrid shearing; mitochondrial RNA polymerase; stem loop