New Insights into the Mechanism of Initial Transcription

Luis E Ramírez-Tapia,Craig T Martin

doi:10.1074/jbc.m112.370643

Luis E Ramírez-Tapia, Craig T Martin

Open Access

https://doi.org/10.1074/jbc.m112.370643

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Abstract

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

Highlights

RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts
The current study aims to understand the energetic forces that underlie initial transcription and abortive cycling by RNA polymerases
The genetically isolated mutation P266L in T7 RNA polymerase presents a valuable tool in understanding the energetics of initial transcription in this model system [8]

Summary

Background

RNA polymerases must couple the energetics of nucleotide addition to the timed release of promoter contacts. During elongation, the active site must maintain a constant sized (at least eight base) RNA-DNA hybrid as the accepting species, dissociating RNA from one end of the hybrid, whereas adding nucleotides to the other Given these different requirements, it is perhaps not surprising that RNA polymerases undergo significant structural rearrangements in transitioning from an initially transcribing complex to a stably elongating complex [2, 3] and that initially transcribing complexes are relatively unstable, producing abortive RNA transcripts, whereas elongation complexes are (and must be) extremely stable during processive elongation [4, 5]. In the model RNA polymerase from bacteriophage T7, a single point mutation (P266L), distant from the hybrid, dramatically reduces the relative production of released abortive transcripts [8], pointing to a more complex process involving the protein. We propose a model for abortive cycling that accounts for this new observation, coupling it to a structural model for the transition to elongation

EXPERIMENTAL PROCEDURES

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DISCUSSION