The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain
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RESEARCH ARTICLE
Open Access
The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain Robert OJ Weinzierl
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.
Background RNA polymerases (RNAPs) play a central role in the regulation of gene expression. Like the majority of the enzymes involved in fundamental biological information-processing functions (for example, replication, transcription, recombination, repair), RNAPs are probably best viewed as intricate molecular machines. The movement of nucleic acid substrates, coupled with various types of active site chemistries, requires a precisely orchestrated sequence of conformational changes of protein domains during the transcription cycle (for recent reviews see [1-4]). The nanomechanical mechanisms guiding the structural rearrangements of domains within the active site are still very poorly understood. Thus far, models of the fundamental reaction catalyzed by RNAPs, the nucleotide addition cycle (NAC), have predominantly been derived from a series of crystal structures that contain RNAPs as apoenzymes (for example [5-9]), or complexed with various Correspondence: [email protected] Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
substrates and inhibitors (for example [10-15]). Such structures, revealing (among other features) pre- and posttranslocation states of RNAPs, have provided the basis for various hypotheses concerning the molecular mechanism of the NAC [1-4,16,17]. There are, however, two potential shortcomings associated with such approaches. First, in order to ‘freeze’ the RNAPs in a crystallizable conformation, substrate analogs or inhibitors need to be chosen that stop the reaction cycle at a specific point. This may result in the adoption of ‘off-pathway’ conformations that do not represent normal enzyme states. A second, more fundamental, problem is that short-lived intermediate structur
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