Bridge Helix Function in RNA Polymerase Catalysis

RNA polymerases are the cellular photocopiers transcribe a temporary copy of a gene ( a transcript ) from the genetic material as required. In the post-genomic era we know the information content stored in our genetic material, but still have lot to learn about the way this information is read out under different biological conditions; RNA polymerases are critically involved in these molecular decision-making events. Although important information about their molecular structure has emerged during the last decade (an achievement which was rewarded with a Nobel Prize to Prof. Roger Kornberg in 2007), many important questions remain unanswered. RNA polymerases can best be imagined as mobile molecular machines that crawl along the DNA to produce their transcripts. In order to do this, RNA polymerases combine complex internal motions with a precisely defined chemical activity (synthesis of the transcript in its catalytic center).
In previous work we identified a key role for a nanomechanical element, the ?Bridge Helix?, located at the core of RNA polymerase. The Bridge Helix carries out its function through changing the settings of two major molecular hinges. Kinking of these molecular hinges changes the arrangement of other structures within the catalytically site to cocordinate their position at various stages of the transcription process. The molecular events surrounding the function of one of these molecular hinges, BH-HN, is the focus of the proposed research. BH-HN is particularly exciting because its existence was only recently discovered and therefore has the potential to cause a significant change in the way we are thinking about RNA polymerase mechanisms and function. We will use newly developed robotic tools to carry out studies in a way that would not be feasible using conventional techniques.
This work is not only of profound scientific interest, but may also give rise to medical applications in the future. Several antibiotics used to fight pathogenic bacteria are known to interfere with the molecular mechanisms of RNA polymerase. In our most recent work we showed that BH-HN appears to be permanently activated in a small number of bacteria, including an emergent pathogen involved in food poisoning and another pathogen with devastatingly lethal effects in East Asia. These examples demonstrate very clearly that results from fundamental sciences often give rise to insights and applications in the real world that have a substantial chance of improving the quality of life and the provision of healthcare.

RNA polymerases (RNAPs) are examples of molecular machines that combine a chemically reactive region with nanomechanical domains to ensure the step-wise movement of the nucleic acid substrates. Although the principles of the chemical reaction mechanism are understood, there are many open questions regarding the coupling of the chemistry with the nanomechanical aspects of the substrates moving through the active site. In previous research I have uncovered several independent lines of evidence in support of conformational changes in the Bridge Helix domain. Certain superactive mutations that stabilize kinked conformations of the Bridge Helix by acting as a molecular switch. One of these molecular hinges, BH-HN is located in the N-terminal region of the Bridge Helix where it is surrounded by other structural elements, such as the beta-D and Link domains. Kinking of BH-HN is likely to result in a considerable spatial redeployment of these two domains, thus coordinating catalysis with substrate translocation. In the proposed work we intend to carry out high-throughput mutagenesis to learn more about the function of the beta-D and Link domains, including how they interact with various N-terminal Bridge Helix conformations. The work will involve innovative robotic approaches and will be supported by fully atomistic molecular dynamics simulations of the domains to establish structure/function relationships.