The Mechanics of Initial Transcription: The RNA-DNA Hybrid Pushes on the Protein, and the Protein Pushes Back!

Abstract

RNA polymerases must couple the energetics of nucleotide addition to drive timed release of strong promoter contacts. RNA polymerases also undergo substantial structural changes in transitioning from sequence-specific initial transcription to stable elongation. Initially transcribing complexes are characteristically unstable, yielding short abortive products, but polymerase mutations have been isolated that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. using fluorescence probes of structural changes, we demonstrate that the low-abortive P266L mutant T7 RNA polymerase also transitions to elongation at longer lengths of RNA. We propose that both properties derive from a weakening of the initial barrier to hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to drive promoter release. While the hybrid pushes on this protein element, the protein in turn pushes back on the hybrid, leading to the observed instability during initial transcription. Parallel biochemical experiments on bacterial RNA polymerase show that the sigma region 3.2 linker (analogous to the eukaryotic RNA polymerase “B-finger”) functions in a very similar manner. Thus the mechanical “pushing” of the RNA-DNA hybrid against the protein is met by a reciprocal, and destabilizing, pushing of the protein against the hybrid.