The availability of in vitro assembly systems to produce recombinant archaeal RNA polymerases (RNAPs) offers one of the most powerful experimental tools for investigating the still relatively poorly understood molecular mechanisms underlying RNAP function. Over the last few years, we pioneered new robot-based high-throughput mutagenesis approaches to study structure/function relationships within various domains surrounding the catalytic center. The Bridge Helix domain, which appears in numerous X-ray structures as a 35-amino-acid-long alpha helix, coordinates the concerted movement of several other domains during catalysis through kinking of two discrete molecular hinges. Mutations affecting these kinking mechanisms have a direct effect on the specific catalytic activity of RNAP and can in some instances more than double it. Molecular dynamics simulations have established themselves as exceptionally useful for providing additional insights and detailed models to explain the underlying structural motions. 1. Introduction RNA polymerases (RNAPs) are key enzymes of the cellular gene expression machineries of all organisms. Despite substantial progress during the last decade in elucidating high-resolution structures of RNAPs and the recent award of a Nobel Prize (Roger Kornberg, Chemistry 2006), there are still many unanswered questions regarding the mechanistic basis of transcription. This is mostly a consequence of the intrinsic complexity of the processes, but also due to a shortage of appropriate experimental data. Current models are predominantly shaped by the interpretation of X-ray crystal structures [1], but such approaches provide only a limited perspective. Crystallization trials require stable, catalytically inactive complexes as starting material, and many short-lived transitory conformations are unlikely to be preserved in crystal structures [2]. During the last decade, we have pioneered alternative experimental strategies based on a hyperthermophilic archaeal system—the euryarchaeon Methanocaldococcus jannaschii—to devise an experimental system capable of generating functional insights in a systematic and high-throughput manner. We succeeded in creating an in vitro transcription system capable of promoter-specific transcription that consists entirely, including the RNAP, of recombinant proteins [3, 4]. Much of this work was guided by the key concept that the archaeal basal transcriptional machinery [5] closely mirrors the core components of the eukaryotic RNA polymerase II (RNAPII) system [6, 7], which is responsible for the highly regulated
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