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首页> 外文期刊>Journal of Molecular Biology >Transcription termination defective mutants of Rho: role of different functions of rho in releasing RNA from the elongation complex
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Transcription termination defective mutants of Rho: role of different functions of rho in releasing RNA from the elongation complex

机译：Rho的转录终止缺陷突变体：rho不同功能在从延伸复合物中释放RNA的作用

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摘要

The transcription termination factor Rho of Escherichia coli is a RNA binding protein which can translocate along the RNA and unwind the RNA:DNA hybrid using the RNA-dependent ATPase activity. In order to investigate the involvement of each of these functions in releasing RNA from the elongation complex, we have isolated different termination defective mutants of Rho by random mutagenesis, characterized them for their different functions and established the structure-function correlations from the available structural data of Rho. These mutations are located within the two domains; the N-terminal RNA binding domain (G51V, G53V, and Y80C and in the C-terminal ATP binding domain (Y274D, P279S, P279L, G324D, N340S, I382N) including the two important structural elements, the Q-loop (P279S, P279L) and R-loop (G324D). Termination defects of the mutants in primary RNA binding domain and Q-loop could not be restored under any conditions that we tested and these were also defective for most of the other functions of Rho. The termination defects of the mutants (Y274D, G324D and N340S), which were mainly defective for secondary RNA binding and likely defective for translocase activity, could be restored under relaxed in vitro conditions. We also show that a mutation in a primary RNA binding domain (Y80C) can cause a defect in ATP binding and induce distinct conformational changes in the distal C-terminal domain, and these allosteric effects are not predictable from the crystal structure. We conclude that the interactions in the primary RNA binding domain and in the Q-loop are mandatory for RNA release to occur and propose that the interactions in the primary RNA binding modulate most of the other functions of Rho allosterically. The rate of ATP hydrolysis regulates the processivity of translocation along the RNA and is directly correlated with the efficiency of RNA release. NusG improves the speed of RNA release and is not involved in any other step. (c) 2007 Elsevier Ltd. All rights reserved.
机译：大肠杆菌的转录终止因子Rho是一种RNA结合蛋白，可以沿着RNA转移并利用RNA依赖性ATPase活性解开RNA：DNA杂种。为了研究这些功能中的每一个参与从延伸复合物中释放RNA的过程，我们通过随机诱变分离了Rho的不同终止缺陷型突变体，对它们的不同功能进行了表征，并从可用的结构数据中建立了结构-功能相关性Rho。这些突变位于两个结构域内。 N端RNA结合结构域（G51V，G53V和Y80C）和C端ATP结合结构域（Y274D，P279S，P279L，G324D，N340S，I382N）包括两个重要的结构元件Q环（P279S， P279L）和R-loop（G324D）。在我们测试的任何条件下，原始RNA结合结构域和Q-loop中突变体的终止缺陷都无法修复，并且对于Rho的其他大部分功能也存在缺陷。突变体（Y274D，G324D和N340S）的缺陷，主要在次级RNA结合方面有缺陷，而在转移酶活性上可能存在缺陷，可以在宽松的体外条件下得到修复。我们还证明了在主要RNA结合域（Y80C）处的突变）可能会导致ATP结合缺陷并在远端C末端结构域中引起明显的构象变化，并且从晶体结构无法预测这些变构效应。我们得出结论，主要RNA结合结构域和Q环中的相互作用一种Rho强制发生RNA释放，并提出一级RNA结合中的相互作用会变构地调节Rho的大多数其他功能。 ATP水解的速率调节了沿RNA转运的过程，并与RNA释放效率直接相关。 NusG提高了RNA释放的速度，并且不参与任何其他步骤。 （c）2007 Elsevier Ltd.保留所有权利。

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