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

雀麦花叶病毒亚基因组启动子的 RNA 重组：与转录的关系

• 批准号: 0317039
• 负责人: Jozef Bujarski
• 金额: --
• 依托单位: Northern Illinois University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0317039&HistoricalAwards=false
• 关键词: RNA; Recombination; Subgenomic; Promoter; Brome

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.

这项研究的长期目标是了解RNA病毒中基因重组的机制。该项目组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 RNA 3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。 本计画将结合活体与离体实验，以剖析sgp介导同源重组的分子机制。 该项目的主要目标是：（1）通过精确定位交叉位点，确定转录起始的作用和Sgp结构的作用，以及鉴定预分离的（+）链，研究体内转录和重组之间的关系;和（ii）通过确定新生链引发的作用来研究体外重组的机制，绘制和关联暂停和结合区域，并将结果与体内结果相关联。 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基因RNA重组的机制，在一个良好的表征模型病毒。这些实验将涉及一种新的重组系统，该系统由转录启动子驱动。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被许多组RNA病毒用于基因表达，但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，以及开发工程化稳定病毒载体的策略。Sgp介导重组的BMV系统是在PI实验室开发的，是植物病毒学中允许研究同源RNA重组机制的独特系统。一般认为，在RNA病毒复制过程中，同源交叉发生频率很高，但却无法检测到。对sgp介导的重组的研究结果将有助于我们了解序列/结构因子在指导同源交叉中的作用。 它们还将增强我们对重叠病毒功能之间在分子水平上的关系的理解。PI还将利用该项目继续对个人进行分子生物学和生物化学原理的培训。 目前在他的实验室进行研究的不仅有本科生、研究生和博士后，还有一名高中教师。