Novel insight into the patterns of in vitro short abortive RNA release by Escherichia coli RNA polymerase

Abstract

E. coli RNA polymerase (RNAP) catalyzes prokaryotic transcription with the σ70 specificity factor, which directs the binding of RNAP to promoter DNA. RNAP operates on the promoter to form an open complex. In transcription initiation, complementary ribonucleotide triphosphates (NTP) bind stepwise to the template DNA in the active site and RNAP catalyzes phosphodiester bond formation. After each NTP addition, the RNA-DNA hybrid translocates into the active site cleft while maintaining promoter contacts. In productive complexes, free energy buildup from translocation allows RNAP to break its specific contacts and escape from the promoter to transcribe full-length RNA. Nonproductive complexes stall before escape, release a short RNA, and re-initiate a cycle of abortive (short RNA) transcription. Dinucleotide RNA is the shortest possible abortive product. Experimental evidence and a thermodynamic rationale are presented indicating that most of the RNA synthesized by nonproductive complexes in vitro for the conditions investigated is 3-mer or longer.