A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter.
We report experiments designed to elucidate the mechanism by which RNA polymerase advances from the open complex to synthesis of a stably bound RNA chain during transcription initiation. Techniques used include deoxyribonuclease I footprinting, methylation protection, and exonuclease III digestion through upstream domains, each applied to the open, abortive and productive transcription complexes of Escherichia coli RNA polymerase with the lac promoter. The results show a slight loss of upstream open complex contacts during abortive transcription of a 6-mer and 8-mer, but a large loss of these contacts upon escape from abortive cycling into productive transcription at the 11-mer. We propose a model for early initiation in which competition between open complex polymerase-DNA contacts on one hand and initiated complex polymerase-DNA-RNA interactions on the other produces a "stressed intermediate" during formation of a short RNA-DNA duplex. The strain energy is relieved either by ejecting the short RNA, resulting in aborted initiation, or by eliminating the sigma subunit and breaking the open complex contacts, thereby escaping abortive cycling into productive transcription. Further evidence for this model is based on the observation that destabilization of interactions specific for either open complex or initiated complex has the predicted effect on the amount of abortive cycling. The model predicts a complicated relationship between overall promoter strength and DNA sequence changes that alter polymerase-DNA interactions.
D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases; D006160:Guanosine Triphosphate; D007293:Inosine Triphosphate; D008745:Methylation; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics*; D013696:Temperature; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D007293:Inosine Triphosphate; D006160:Guanosine Triphosphate; D012321:DNA-Directed RNA Polymerases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D003850:Deoxyribonuclease I