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首页> 美国卫生研究院文献>other >Probing DNA Binding DNA Opening and Assembly of a Downstream Clamp/Jaw in E. coli RNA Polymerase – λPR Promoter Complexes Using Salt and the Physiological Anion Glutamate
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Probing DNA Binding DNA Opening and Assembly of a Downstream Clamp/Jaw in E. coli RNA Polymerase – λPR Promoter Complexes Using Salt and the Physiological Anion Glutamate

机译：探测DNa结合一个下游夹紧装置/颚在大肠杆菌RNa聚合酶的DNa打开和装配 - λpR启动子配合物使用盐和生理阴离子谷氨酸

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摘要

Transcription by all RNA polymerases (RNAP) requires a series of large-scale conformational changes to form the transcriptionally-competent open complex RPo. At the λPR promoter, E. coli σ70 RNAP first forms a wrapped, closed 100 bp complex I1. The subsequent step opens the entire 13 base DNA bubble, creating the relatively unstable (open) complex I2. Additional conformational changes convert I2 to the stable RPo. Here we probe these events by dissecting the effects of Na+ salts of Glu−, F− and Cl− on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I1 at 25 °C is ~30-fold larger in Glu− than in Cl− at the same [Na+], with the same log-log [salt] dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I1 to I2) are only weakly [salt]-dependent, and the opening rate constant is insensitive to replacement of Cl− by Glu−. These very small effects of [salt] on a process (DNA opening) which is strongly [salt]-dependent in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process, and/or that compensation is present between ion uptake and release.Replacement of Cl− by Glu− or F− at 25 °C greatly increases the lifetime of RPo and greatly reduces its [salt]-dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu− and F− drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I2 to RPo, while Cl− does not. Because the Hofmeister effect of these anions largely compensates for the destabilizing coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RPo remains long-lived even at 0.5 M Na+ in Glu− or F− salts. The observation that Eσ70 RPo are exceedingly long-lived in moderate to high [Glu−] argues that Eσ70 RNAP do not dissociate from strong promoters in vivo when the cytoplamsic increases during osmotic stress.

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