Bridge helix and trigger loop perturbations generate superactive RNA polymerases

<p>Abstract</p> <p>Background</p> <p>Cellular 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.</p> <p>Results</p> <p>Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the <it>Methanocaldococcus jannaschii </it>RNA polymerase A' subunit, revealing a wide spectrum of <it>in vitro </it>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.</p> <p>Conclusion</p> <p>The 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.</p

The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain

Abstract Background Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-HC) plays a critical role in controlling the catalytic rate. Results Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-HN) is presented. The nanomechanical properties of BH-HN emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-HN displays functional properties that are distinct from BH-HC, suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms. Conclusions The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.</p

Revealing the functions of TFIIB

The TFIIB linker domain stimulates the catalytic activity of archaeal RNAP. By characterizing a range of super-stimulating mutants, we identified a novel rate-limiting step in transcription initiation. Our results help to interpret structural findings and pave the way toward higher-resolution structures of the RNAP-TFIIB linker interface