Wang Sheng, Mortazavi Leyla, White K Andrew
Department of Biology, York University, Toronto, Ontario, Canada.
J Virol. 2008 Apr;82(8):3864-71. doi: 10.1128/JVI.02416-07. Epub 2008 Feb 6.
Subgenomic (sg) mRNAs are small viral messages that are synthesized by polycistronic positive-strand RNA viruses to allow for the translation of certain viral proteins. Tombusviruses synthesize two such sg mRNAs via a premature termination mechanism. This transcriptional process involves the viral RNA-dependent RNA polymerase terminating minus-strand RNA synthesis prematurely at internal RNA signals during copying of the viral genome. The 3'-truncated minus-strand RNAs generated by the termination events then serve as templates for sg mRNA transcription. A higher-order RNA structure in the viral genome, located just upstream from the termination site, is a critical component of the RNA-based polymerase attenuation signal. Here, we have analyzed the role of this RNA structure in mediating efficient sg mRNA2 transcription. Our results include the following: (i) we define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) we show that the distribution of stability within an attenuation structure affects its function; (iii) we establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) we prove that the higher-order RNA structure forms and functions in the plus strand; (v) we provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; (vi) we demonstrate that sg mRNA transcription can be controlled artificially through small-molecule activation using RNA aptamer technology. These findings provide important new insights into the premature termination mechanism and present a novel approach to regulate the transcriptional process.
亚基因组（sg）mRNA是由多顺反子正链RNA病毒合成的小病毒信使，用于某些病毒蛋白的翻译。番茄丛矮病毒通过提前终止机制合成两种这样的sg mRNA。这个转录过程涉及病毒RNA依赖性RNA聚合酶在病毒基因组复制过程中在内部RNA信号处提前终止负链RNA合成。终止事件产生的3'端截短的负链RNA随后作为sg mRNA转录的模板。病毒基因组中位于终止位点上游的高阶RNA结构是基于RNA的聚合酶衰减信号的关键组成部分。在这里，我们分析了这种RNA结构在介导高效sg mRNA2转录中的作用。我们的结果如下：（i）我们定义了一个可操作的高阶RNA衰减结构所需的最小整体热力学稳定性；（ii）我们表明衰减结构内稳定性的分布会影响其功能；（iii）我们确定RNA四链体结构可以作为一种有效的衰减结构；（iv）我们证明高阶RNA结构在正链中形成并发挥功能；（v）我们提供证据表明转录不需要特定的衰减结构结合蛋白因子；（vi）我们证明可以使用RNA适体技术通过小分子激活来人工控制sg mRNA转录。这些发现为提前终止机制提供了重要的新见解，并提出了一种调节转录过程的新方法。
