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首页> 外文期刊>Journal of Molecular Biology >ANALYSIS OF E. COLI RHO FACTOR - MUTATIONS AFFECTING SECONDARY-SITE INTERACTIONS
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ANALYSIS OF E. COLI RHO FACTOR - MUTATIONS AFFECTING SECONDARY-SITE INTERACTIONS

机译：E. COLI RHO因子-影响次生相互作用的突变分析

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To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1. They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235). Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in secondary-site RNA activation, with K-m values for r(C)(10) of 100 mu M and similar to 650 mu M, respectively, compared to the wild-type value of 4 mu M. These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro. By contrast, PH235 was very efficient at interacting with r(C)io at the secondary site, with a measured K-m of 0.5 mu M, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination. (C) 1995 Academic Press Limited [References: 45]
机译：为了定义和区分rho蛋白的线性序列内的一级和二级RNA结合位点，我们研究了两个突变等位基因，rho-115和rhosuA1。它们首先被鉴定为体内转录终止有缺陷，后来被证明分别与一级和二级位点的RNA相互作用存在缺陷。 rhosuA1的测序揭示了在352位（KE352）上一个赖氨酸变为谷氨酸残基的变化，而rho-115则带有两个突变，甘氨酸99为缬氨酸（GV99）和脯氨酸235为组氨酸（PH235）。纯化了在这三个位置的每个位置带有单个突变的蛋白质，并与野生型蛋白质进行了比较。我们发现KE352和GV99在次级位点RNA激活中均存在缺陷，与野生型4μm的r（C）（10）的Km值分别为100μM和650μM相似。 M.这些观察到的次级位点缺陷与降低的解旋酶和ATPase活性以及体外转录终止活性的丧失有关。相比之下，PH235在次级位点与r（C）io相互作用非常有效，测量的K-m为0.5μM，并且通过其ATPase，解旋酶和终止能力可以判断其具有高活性rho的特征。我们的结果表明，多肽中三个非常不同的位置处的突变会影响RNA的二级位点激活，并且这些相互作用在ATP水解，解旋酶活性和转录终止中起关键作用。 （C）1995 Academic Press Limited [参考号：45]

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