Insights into Abortive Cycling during Initial Transcription by T7 RNA Polymerase

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RNA polymerases are characterized by conflicting requirements during initial transcription and elongation. In the initial phase, an RNA polymerase must recognize a specific promoter sequence, melt the DNA at the start site, initiate de novo synthesis of a dimer RNA, and extend that RNA in a hybrid that is thermodynamically unstable to collapse of the melted bubble. Hybrid growth in turn ultimately drives promoter release and a structural transition that enables topologically locked RNA entry into a stabilizing exit channel, establishing a stable elongation complex.Perhaps not surprisingly, short RNAs are not stable during initial transcription, leading to their abortive release. But hybrid stability is only part of the story. In T7 RNA polymerase, a single point mutation (P266L) in the protein, distant from the hybrid, leads to a dramatic reduction in abortive release on most promoters. Current models suggest that the mutant releases promoter contacts more readily, transitioning to a stable elongation complex sooner.New results reveal the opposite behavior: P266L releases promoter contacts later than wild type, suggesting an overall increase in stability of the initially transcribing complex. Based on kinetic studies, we present a testable model to explain how growth of the hybrid serves to drive a timed transition to elongation.