An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations
An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations
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DOI：
10.1128/jvi.01129-16
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发表时间：
2016-10-01
影响因子：
5.4
通讯作者：
Simon, Anne E.
中科院分区：
文献类型：
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作者：
Kuhlmann, Micki M.;Chattopadhyay, Maitreyi;Simon, Anne E.
核糖体重新编码被RNA病毒用来翻译通读或移码过去的终止密码子以合成延伸产物。重新编码位点以及下游的重新编码刺激元件(RSE)长期以来一直被研究在报告结构中，因为这些片段本身就调节了惯常的重新编码水平，因此被认为包含建立适当的终止与重新编码比率的完整指令。来自Tombus病毒科和Luteo-Virridae的RSE被认为是例外，因为它们包含一个与3‘端长距离的RNA-RNA连接。这种相互作用被认为是用来替代假结节的，而假结节被认为是在托姆斯维里RSE中缺失的。我们提供的证据表明，CarmoVirus Turnip crinkle Virus(TCV)在系统发育上保守的RSE采用了一种替代的较小的结构，该结构延伸了上游保守的发夹，并且这种替代结构是植物细胞中新生病毒RNA和在体外合成RNA时RSE的主要形式。TCV RSE还含有一个与远距离相互作用的内部伪结，该伪结与系统发育保守结构不相容。刚过重新编码位点的保守残基对重新编码很重要，这些残基也在伽马逆转录病毒的RSE中保守。我们的数据证明了TCV RSE的动态性质，并表明使用报告结构的研究可能不能有效地概括RSE介导的病毒基因组内的重新编码。重要的核糖体重新编码被RNA病毒用来使核糖体能够延长翻译超过终止密码子以合成更长的产物。当被切除并放置在报告构建体中时，重新编码位点和下游重新编码刺激元件(RSE)调节预期的重新编码水平，因此被认为包含建立终止与重新编码的适当比率的完整指令。我们提供的证据表明，大多数TCV RSE采用了一种延伸上游保守发夹的替代结构，这种替代结构而不是系统发育保守结构，是体外合成和植物细胞中RSE的主要形式。TCV RSE还含有与系统发育保守结构不相容的内部假结和通往3‘端的RNA桥。这些数据表明，TCV RSE在结构上是动态的，可能需要多种构象来调节核糖体的通读。
Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteo-viridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3' end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes.IMPORTANCERibosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin and that this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3' end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough.