tRNA Genes and Genetic Mobility in S. cerevisiae

转移RNA基因（tDNA）不仅对它们编码的RNA很重要，而且对它们在基因组功能和进化中的作用也很重要。 它们被认为是异染色质DNA的边界、pol II转录的阻遏物、基因组重排和异位重组的位点、致病岛插入的位点以及长末端重复序列（LTR）逆转录转座子整合的靶点。 Sandmeyer实验室研究Ty3，这是酿酒酵母中的一种逆转录病毒样元件，插入RNA聚合酶III转录起始位点。从包括病毒样颗粒和质粒携带的靶标的体外系统已知，与DNA结合的pol III转录因子TFIIIB足以进行体外整合。然而，关于这个元素，tDNA和它们的基因组背景之间的关系还有很多东西要了解。 该项目有三个目标：1）Ty3整合前复合物（PIC）的表征和确定的体外Ty3整合系统的重构。Ty3生命周期中的中心步骤是将VLP转化为整合能力cDNA相关的PIC。将基于cDNA整合活性分离PIC组分。它将被表征为Ty3和宿主蛋白质，并使用AFM成像。 2)鉴定Ty3 PIC与其靶标之间的特异性相互作用。Ty3体外整合的最低靶标要求由TATA结合蛋白（TBP）和与RNA pol III启动子结合的Brf 1组成。 PIC和靶标中相互作用的结构域将通过检测Ty3蛋白（特别是整合酶）与Brf 1之间的相互作用来鉴定。 如果这些候选物不相互作用，则将对Ty3进行诱变，并使用遗传筛选来鉴定位置特异性受影响的突变体。 3)Ty3整合的染色体靶标特征的表征。 将使用Pol III转录基因的不同子集来解决体内基于染色体的转录活性和Ty3靶向的关系。 将分别使用染色质免疫沉淀定量PCR监测TFIIIB和TFIIIC以及Pol III占用率和转座。 反转录转座子和tRNA基因是影响原核和真核基因组动员的主要力量，但实际上对tRNA基因作为染色体区域分隔符的能力或为什么反转录转座子更喜欢整合到某些区域而不是其他区域的能力了解相对较少。 这项研究将导致更好地了解这两个相互作用和动态的基因组成分。 PI还将使用它向暑期高中生，本科生物学专业学生和研究生介绍酵母分子生物学和基因组研究的原理。 UCI有一个活跃的少数民族科学家计划，来自各种教育机构的学生，包括社区学院和医学院，都被招募到这个计划中，并有资格参加这个项目。 数据将通过出版物和在公共网站上张贴总结调查结果的数据库来传播。

Transfer RNA genes (tDNA's) are important not only for the RNA's they encode, but also for their roles in genomic function and evolution. They have been identified as boundaries of heterochromatic DNA, repressors of pol II transcription, sites of genomic rearrangement and ectopic recombination, sites of pathogenic island insertion, and targets of long terminal repeat (LTR) retrotransposon integration. The Sandmeyer laboratory studies Ty3, a retroviruslike element in Saccharomyces cerevisiae that inserts at RNA polymerase III transcription initiation sites. It is known from an in vitro system that includes viruslike particles and a plasmid-borne target, that the pol III transcription factor TFIIIB bound to DNA is sufficient for integration in vitro. However, much remains to be learned about the relationship between this element, tDNA's, and their genomic contexts. This project has three goals: 1) Characterization of the Ty3 preintegration complex (PIC) and reconstitution of a defined in vitro Ty3 integration system. A central step in the Ty3 lifecycle is conversion of the VLP into the integration-competent cDNA-associated PIC. A PIC fraction will be isolated based on cDNA integrating activity. It will be characterized for Ty3 and host proteins and imaged using AFM. 2) Identification of specific interactions between the Ty3 PIC and its target. The minimal target requirement for in vitro integration of Ty3 is comprised of the TATA binding protein (TBP) and Brf1 bound to a RNA pol III promoter. The domains in the PIC and target which interact will be identified by testing for interactions between Ty3 proteins, particularly integrase, and Brf1. If those candidates do not interact, Ty3 will be mutagenized and a genetic screen will be used to identify mutants affected in position specificity. 3) Characterization of features of chromosomal targets of Ty3 integration. The relationship of in vivo chromosome-based transcriptional activity and Ty3 targeting will be addressed using a diverse subset of Pol III-transcribed genes. TFIIIB and TFIIIC and Pol III occupancy and transposition will be monitored using chromatin immunoprecipitation quantitative PCR, respectively. Retrotransposons and genes for tRNA's are major forces affecting mobilization in prokaryotic and eukaryotic genomes yet relatively little is actually understood about the ability of tRNA genes to act as delimiters of chromosomal regions or why retrotransposons prefer integration in some regions over others. This research will lead to a better understanding of these two interacting and dynamic genomic components. It will also be used by the PI to introduce summer high school students, undergraduate biology majors, and graduate students to the principles of yeast molecular biology and genomic research. UCI has an active Minority Scientist Program, and students from a wide range of educational institutions, including community colleges and medical schools, are recruited into this program and are eligible for this project. Data will be disseminated by publication and by posting a database summarizing the findings at a public website.