Transcription Elongation Complex Stability

Xiaoqing Liu,Craig T Martin

doi:10.1074/jbc.m109.056820

Abstract

Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi- and single subunit RNA polymerases have evolved an 8-10-base pair RNA-DNA hybrid as a part of a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4-base pair hybrid can nevertheless be more stable than those with the normal 8-base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.

Highlights

RNA polymerases are inherently processive in that, unlike distributive DNA polymerases, an incompletely synthesized RNA cannot be further extended by the binding and action of a second polymerase
The stability of a halted elongation complex depends on numerous factors, but it has been recently demonstrated that dissociation occurs primarily via a mechanism in which forward translocation of the complex leads to a shortening of the RNA-DNA hybrid (11, 13)
This supports an alternate explanation for stability: a topological lock in which the 3Ј end of the RNA is bound at the active site, with the RNA wrapping around the template strand and through the RNA exit channel (11, 15)

Summary

Introduction

RNA polymerases are inherently processive in that, unlike distributive DNA polymerases, an incompletely synthesized RNA cannot be further extended by the binding and action of a second polymerase. Note that in these experiments, ATP is depleted within 10 min; reactions with lower concentrations of enzyme and DNA yield still longer average transcript lengths.3 incorporation of an upstream mismatch within the bubble has been reported to increase the stability of halted elongation complexes (11) and, as expected, yields longer slippage products.

Results

Conclusion