Bridge helix and trigger loop perturbations generate superactive RNA polymerases

Lin Tan,Hannah C Carney,Simone Wiesler,Robert Oj Weinzierl,Dominika Trzaska

doi:10.1186/jbiol98

Abstract

BackgroundCellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available.ResultsUsing a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A' subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop.ConclusionThe results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis.

Highlights

Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site
Bridge helix mutants display a broad spectrum of catalytic activity phenotypes The bridge helix of M. jannaschii RNA polymerases (RNAPs) is located near the carboxyl terminus of the mjA′ subunit and is clearly identifiable by its colinearity and high degree of sequence identity and/or similarity to bacterial and eukaryotic orthologs [25] (Figure 1b)
The resulting RNAP variants were initially screened using a high-throughput trichloroacetic acid (TCA) precipitation assay that measures the incorporation of [32P]rUTP into transcripts using nuclease-activated DNA as template

Summary

Introduction

Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. The bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. Periodic conversions from the straight to the various kinked bridge helix conformations during each ribonucleotide addition step could, in principle, provide a mechanical basis for translocating the nucleic acid substrates through the active site in single nucleotide steps [5,6,14,15] (Figure 1a,c). Recent models emphasize a direct role for the trigger loop in controlling the catalytic functions of RNAPs through conformation-specific contacts with the NTP in the nucleotide insertion site [7,8,18]. The crucial role of the combined bridge helix/trigger loop mechanism in RNAP function is most clearly demonstrated by the inhibitory action of bacterial antibiotics and eukaryotic toxins that block bridge helix and trigger loop movements [12,13, 19,20,21] (Figure 1b)

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Results

Discussion

Conclusion