RNAPs (RNA polymerases) are complex molecular machines containing structural domains that co-ordinate the movement of nucleic acid and nucleotide substrates through the catalytic site. X-ray images of bacterial, archaeal and eukaryotic RNAPs have provided a wealth of structural detail over the last decade, but many mechanistic features can only be derived indirectly from such structures. We have therefore implemented a robotic high-throughput structure–function experimental system based on the automatic generation and assaying of hundreds of site-directed mutants in the archaeal RNAP from Methanocaldococcus jannaschii. In the present paper, I focus on recent insights obtained from applying this experimental strategy to the bridge–helix domain. Our work demonstrates that the bridge–helix undergoes substantial conformational changes within a narrowly confined region (mjA′ Ala822-Gln823-Ser824) during the nucleotide-addition cycle. Naturally occurring radical sequence variations in plant RNAP IV and V enzymes map to this region. In addition, many mutations within this domain cause a substantial increase in the RNAP catalytic activity (‘superactivity’), suggesting that the RNAP active site is conformationally constrained.
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Conference Article| March 22 2010
Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases
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Robert O.J. Weinzierl; Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases. Biochem Soc Trans 1 April 2010; 38 (2): 428–432. doi: https://doi.org/10.1042/BST0380428
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