Chalissery Jisha, Banerjee Sharmistha, Bandey Irfan, Sen Ranjan
Laboratory of Transcription Biology, Centre for DNA Fingerprinting and Diagnostics, ECIL Road, Nacharam, Hyderabad-500076, India.
J Mol Biol. 2007 Aug 24;371(4):855-72. doi: 10.1016/j.jmb.2007.06.013. Epub 2007 Jun 9.
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.
大肠杆菌的转录终止因子Rho是一种RNA结合蛋白，它能够沿着RNA移位，并利用RNA依赖性ATP酶活性解开RNA:DNA杂交链。为了研究这些功能中的每一项在从延伸复合物中释放RNA过程中的作用，我们通过随机诱变分离出了不同的Rho终止缺陷突变体，对它们的不同功能进行了表征，并根据Rho的现有结构数据建立了结构-功能相关性。这些突变位于两个结构域内；N端RNA结合结构域（G51V、G53V和Y80C）以及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环（G324D）。在我们测试的任何条件下，初级RNA结合结构域和Q环中突变体的终止缺陷都无法恢复，并且它们在Rho的大多数其他功能方面也存在缺陷。主要在二级RNA结合方面存在缺陷且可能在转位酶活性方面存在缺陷的突变体（Y274D、G324D和N340S）的终止缺陷，在宽松的体外条件下可以恢复。我们还表明，初级RNA结合结构域中的一个突变（Y80C）可导致ATP结合缺陷，并在远端C端结构域中诱导明显的构象变化，而这些变构效应无法从晶体结构中预测。我们得出结论，初级RNA结合结构域和Q环中的相互作用对于RNA释放的发生是必不可少的，并提出初级RNA结合中的相互作用通过变构调节Rho的大多数其他功能。ATP水解速率调节沿着RNA的转位持续性，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。
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