RNA Recombination at the Subgenomic Promoter in Brome Mosaic Virus: Relationship with Transcription

这项研究的长期目标是了解RNA病毒基因重组的机制。该项目团队先前描述了雀麦花叶病毒（BMV） RNA（一种模型+链RNA病毒）3'非编码区内的节段间重组。最近，该团队在BMV RNA3的亚基因组启动子（sgp）区域绘制了RNA重组热点，并证明了RNA重组与转录活性之间的关系。为了剖析sgp介导的同源重组的分子机制，本项目将包括体内和体外实验。该项目的主要目标是：(1)通过精确定位交叉位点，研究体内转录与重组之间的关系，确定转录起始和sgp结构的作用，鉴定预分离（+）链；（ii）通过确定新生链启动的作用，绘制和关联暂停区和结合区，并将结果与体内的结果联系起来，研究体外重组的机制。这些研究代表了一种新的分子方法，将有助于研究人员在分子水平上了解具有良好特征的模型病毒中遗传RNA重组的机制。实验将涉及一个由转录启动子驱动的新的重组系统。研究人员将确定重组和转录是否利用相同的RNA信号，以及新生链是否可以引发重组。虽然sgp被许多RNA病毒群用于基因表达，但研究结果可以扩展到理解不使用sgp进行基因表达的RNA病毒中的RNA重组，以及开发设计稳定病毒载体的策略。由PI实验室开发的sgp介导重组的BMV系统是植物病毒学中一个独特的系统，可以研究同源RNA重组的机制。人们普遍认为，在RNA病毒复制过程中，同源交叉发生的频率很高，但会逃避检测。sgp介导重组的研究结果将有助于我们了解序列/结构因素在指导同源交叉中的作用。它们也将增强我们在分子水平上对重叠病毒功能之间关系的理解。PI还将利用该项目继续培训分子生物学和生物化学原理方面的个人。目前在他实验室进行研究的既有本科生、研究生和博士后，也有一名高中教师。

The long-term goal of this research is to understand the mechanism(s) of genetic recombination in RNA viruses. The project team has previously described inter-segmental recombination within the 3' noncoding region of brome mosaic virus (BMV) RNAs, a model + strand RNA virus. More recently, the team has mapped an RNA recombination hot spot at the subgenomic promoter (sgp) region of BMV RNA3, and demonstrated a relationship between RNA recombination and transcriptional activity. To dissect the molecular mechanism of sgp-mediated homologous recombination, this project will involve both in vivo and in vitro experiments. The project focuses on the following goals: (i) study the relation between transcription and recombination in vivo through precise mapping of crossover sites, determining the role of initiation of transcription and that of sgp structure and identification of pre-detached (+) strands; and (ii) study the mechanism of recombination in vitro through determining the role of priming by nascent strands, mapping and correlating pausing and binding regions and relating the results to those in vivo. These studies represent a novel molecular approach that will help the researchers to understand the mechanism of genetic RNA recombination at the molecular level in a well-characterized model virus. The experiments will concern a new recombination system that is driven by a promoter of transcription. The investigators will determine whether recombination and transcription utilize the same RNA signals and if nascent strands can prime recombination. Although the sgp is used by many groups of RNA viruses for gene expression, the results can be extended to the understanding of RNA recombination in RNA viruses that do not use sgps for gene expression, as well as in developing strategies to engineer stable viral vectors. The BMV system of sgp-mediated recombination, developed in the PI's laboratory, is a unique system in plant virology that permits studies on the mechanism of homologous RNA recombination. A general belief exists that homologous crossovers occur at high frequency during replication of RNA viruses but escape detection. The results of the studies on the sgp-mediated recombination will contribute to our knowledge about the role of sequence/structure factors in directing homologous crossovers. They will also enhance our understanding of the relation between overlapping viral functions at the molecular level. The PI will also use this project to continue training individuals in the principles of molecular biology and biochemistry. Those currently carrying out research in his laboratory include not only undergraduate, graduate, and postdoctoral students, but also a high-school teacher.