Effects of reaction conditions on RNA secondary structure and on the helicase activity of Escherichia coli transcription termination factor Rho.
Effects of reaction conditions on RNA secondary structure and on the helicase activity of Escherichia coli transcription termination factor Rho.
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反应条件对RNA二级结构和大肠杆菌转录终止因子Rho解旋酶活性的影响。
DOI：
10.1006/jmbi.1998.1814
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
vonHippel,PH
中科院分区：
文献类型：
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作者：
Walstrom,KM;Dozono,JM;vonHippel,PH
大肠杆菌转录终止蛋白 rho 的 ATP 酶和解旋酶活性已在多种改变其转录终止活性的反应条件下进行了研究。这些条件包括 50 至 150 mM 的 KCl、KOAc 或 KGlu 浓度以及 1 至 5 mM 的 Mg(OAc)2 浓度（在 1 mM ATP 存在下）。在较高的 KCl 或较高的 Mg(OAc)2 浓度下，我们发现 rho 六聚体沿 RNA 的易位比在 50 mM 单价盐浓度和 1 mM Mg(OAc)2 下测量的相同过程更慢且持续性更差。在减慢易位的反应条件下，rho 的 ATP 酶活性也会降低。 RNA 熔解实验表明，随着 KCl 或 Mg(OAc)2 浓度的增加，rho 的 ATP 酶活性降低，解旋酶活性减慢，同时解旋酶亚态的 RNA 部分的二级结构也随之增加。相比之下，在聚(rC)（一种不形成盐浓度依赖性二级结构的合成RNA）存在下，rho 的 ATP 酶活性在三种单价盐中表现出相同。因此，盐不会直接影响rho蛋白的结构或构象或rho与单链RNA的结合。然而，rho 沿 RNA 的易位在 150 mM KOAc 或 KGlu 中比在 150 mM KCl 中进行性更强，而 RNA 二级结构在所有三种单价盐中都是相同的。因此，当RNA底物能够形成二级结构时，反应中存在的一价盐可能直接影响rho-RNA相互作用。使用不包含 rho 加载位点的 RNA 分子进行的解旋酶实验表明，rho 沿这种潜在解旋酶底物的易位程度较低。这些结果表明rho的解旋酶活性可能受到RNA二级结构的显着调节。此外，将 rho 活性集中在含有非结构化 rho 加载位点的转录本上的机制之一可能是，沿着此类分子的 rho 易位比沿着细胞中更结构化的 RNA 分子的进行性更强。
The ATPase and helicase activities of the Escherichia coli transcription termination protein rho have been studied under a variety of reaction conditions that alter its transcription termination activity. These conditions include KCl, KOAc, or KGlu concentrations from 50 to 150 mM and Mg(OAc)2concentrations from 1 to 5 mM (in the presence of 1 mM ATP). In higher KCl or higher Mg(OAc)2concentrations we found that the translocation of rho hexamers along RNA was slower and less processive than the same process measured at 50 mM monovalent salt concentrations and 1 mM Mg(OAc)2. The ATPase activity of rho was also decreased under reaction conditions that slowed translocation. RNA melting experiments showed that the decreased ATPase activity of rho and the slower helicase activity at increased KCl or Mg(OAc)2concentrations are accompanied by a concomitant increase in the secondary structure of the RNA portion of the helicase substate. In contrast, the ATPase activity of rho in the presence of poly(rC), a synthetic RNA that does not form salt-concentration-dependent secondary structure, was shown to be the same in each of the three monovalent salts. Thus, the salts do not directly affect the structure or conformation of the rho protein or the binding of rho to single-stranded RNA. However, the translocation of rho along RNA was more processive in 150 mM KOAc or KGlu than in 150 mM KCl, while the RNA secondary structure was the same in all three monovalent salts. Therefore, the monovalent salt present in the reaction may directly affect rho-RNA interactions when the RNA substrate can form secondary structure. Helicase experiments with an RNA molecule that does not contain a rho loading-site showed that rho translocates less processively along this potential helicase substrate. These results suggest that the helicase activity of rho may be significantly regulated by RNA secondary structure. In addition, one of the mechanisms to concentrate the activity of rho on transcripts containing unstructured rho loading sites may be that rho translocation along such molecules is more processive than it is along more structured RNA molecules in the cell.