Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases.
Nanomechanical constraints acting on the catalytic site of cellular RNA polymerases.
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作用于细胞 RNA 聚合酶催化位点的纳米力学约束。
DOI：
10.1042/bst0380428
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发表时间：
2010
影响因子：
3.9
通讯作者：
Weinzierl RO
中科院分区：
文献类型：
--
作者：
Weinzierl RO
RNAP（RNA聚合酶）是复杂的分子机器，其包含协调核酸和核苷酸底物通过催化位点的运动的结构域。在过去的十年中，细菌、古细菌和真核生物RNAP的X射线图像提供了丰富的结构细节，但许多机械特征只能间接地从这些结构中获得。因此，我们已经实现了一个机器人高通量的结构-功能实验系统的基础上，自动生成和分析数百个定点突变体的古细菌RNAP从詹氏甲烷球菌。在本文中，我专注于最近的见解，从应用这种实验策略的桥梁螺旋结构域。我们的工作表明，在核苷酸添加循环中，桥螺旋在狭窄的区域（mjA′ Ala 822-Gln 823-Ser 824）内经历了实质性的构象变化。植物RNAP IV和V酶中天然存在的自由基序列变异映射到该区域。此外，该结构域内的许多突变导致RNAP催化活性的大幅增加（“超活性”），表明RNAP活性位点在构象上受到限制。
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 fromMethanocaldococcus 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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