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首页> 外文期刊>Journal of Molecular Biology >Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.
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Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.

机译：与新生RNA的相互作用是在体外将Rho募集到转录延伸复合物中的前提条件。

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In the conventional model of the Rho-dependent transcription termination, the terminator Rho binds to the rut (Rho utilization) site and translocates along the nascent RNA prior to making possible interactions with the elongating RNA polymerase (RNAP). Even though the interaction between Rho and isolated RNAs was studied in great detail, the same has never been shown with the nascent RNA emerging from the transcription elongation complex (EC). Direct demonstration and requirement of the Rho-nascent RNA binding become even more important because of the recently proposed alternative model where Rho loads onto the RNAP prior to the formation of the nascent RNA. Here, we have measured the direct association of Rho in vitro with the free RNAP, RNAP-promoter binary complex and stalled ECs with varied length of RNA. We observed the association of Rho only with the ECs having the rut-site-containing long nascent RNA. This association was significantly reduced when either a Rho mutant, Y80C, defective for RNA binding or an antisense oligo to the rut site was used or when the rut site was eliminated by RNase digestion or replacement with a random RNA sequence. The presence of EC-bound NusG, the binding partner of Rho, did not facilitate this association. RNase footprinting of the Rho-EC complex revealed a clear Rho-mediated protection of the rut sites on the nascent RNA. We concluded that the nascent RNA loading of Rho and its interaction with the rut site are mandatory and prerequisites for its recruitment to the EC under in vitro experimental conditions.
机译：在Rho依赖性转录终止的常规模型中，终止子Rho结合到rut（Rho利用）位点，并沿着新生的RNA转移，然后才可能与延伸的RNA聚合酶（RNAP）相互作用。尽管对Rho和分离的RNA之间的相互作用进行了详细的研究，但从未发现转录延伸复合物（EC）产生的新生RNA会发生同样的情况。由于最近提出的替代模型（Rho在新生RNA形成之前先加载到RNAP上），对Rho新生RNA结合的直接证明和要求变得更加重要。在这里，我们已经测量了Rho在体外与游离RNAP，RNAP启动子二元复合物和具有不同长度RNA的停滞EC的直接关联。我们观察到Rho仅与具有含车辙位点的长新生RNA的EC关联。当使用Rho突变体Y80C（对于RNA结合有缺陷）或反义寡核苷酸对rut位点时，或者通过RNase消化或用随机RNA序列置换消除了rut位点时，这种关联会大大降低。 EC结合的NusG（Rho的结合伴侣）的存在并不促进这种结合。 Rho-EC复合物的RNase足迹揭示了Rho介导的对新生RNA上的车辙位点的清晰保护。我们得出的结论是，在体外实验条件下，Rho的新生RNA负载及其与rut位点的相互作用是强制性的，也是其募集到EC的前提。

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