Analysis of Termination Mutants of RNA Polymerase II

该项目将结合遗传学和生物化学研究RNA聚合酶（Pol）II对酵母酿酒酵母中蛋白质编码基因3'末端加工和下游终止机制的贡献。终止-从DNA中释放转录聚合酶复合物-是Pol II转录周期中的重要事件，但了解甚少。在真核细胞核中，成熟mRNA的3'末端通过切割和聚腺苷酸化产生。经过加工位点的转录发出信号，使Pol II延伸复合物终止于DNA的下游位置。转录物切割和多聚腺苷酸化所需的许多蛋白质已被鉴定和表征，但将这些事件与终止偶联的机制还不清楚。该项目将集中在Pol II在多聚腺苷酸化和终止偶联中的作用，通过表征在终止缺陷筛选中分离的酵母Pol II的第一和第二大亚基的突变变体。这组突变确定了酶表面上的重要残基的新簇，这些残基可能是其他蛋白质的相互作用位点。该项目的一个具体目标是识别结合这些位点的蛋白质伴侣。标准方法，包括酵母双杂交方法和抑制筛选将用于解决这一目标。 其他突变改变了接近延伸复合物中的核酸的残基，因此更可能影响对终止和/或聚腺苷酸化信号的响应重要的内在Pol II特性。第二个具体的目的是调查的机制，潜在的终止缺陷的每一类突变体，在体外和功能测试在体内使用的组合的生化测定。总之，这些实验的结果将提供对Pol II的伸长特性响应于3'末端加工事件而发生的急剧变化的潜在机制的更详细的理解。RNA聚合酶将存储在DNA中的遗传信息转录成信使RNA，然后将其用作蛋白质合成的模板。这些酶是复杂的机器，必须识别每个基因侧翼的编码信号，并指定转录应该开始和停止的位置。这项研究将深入了解来自模式生物的RNA聚合酶使用编码信息完成和释放RNA，然后从DNA上脱落，再循环到另一个基因的机制。提供适合本科生培养的资源和项目是实验设计中的重要考虑因素。虽然有些实验更适合研究生，但该项目的其他方面特别适合本科生的经验和时间限制。首席研究员的研究小组在指导本科生方面有着良好的记录，其中许多人已经在学术界和工业界做出了未来的科学贡献和职业生涯。

The project will be a combined genetic and biochemical investigation of the contribution of RNA polymerase (Pol) II to the mechanism of 3' end processing and termination downstream of protein-encoding genes in the yeast Saccharomyces cerevisiae. Termination - the release of the transcribing polymerase complex from the DNA - is an essential, but poorly understood, event in the Pol II transcription cycle. In eukaryotic nuclei, the 3' ends of mature mRNAs are generated by cleavage and polyadenylation. Transcription past the site at which processing occurs signals the Pol II elongation complex to terminate at downstream positions on the DNA. Many proteins required for transcript cleavage and polyadenylation have been identified and characterized, but the mechanism(s) that couple these events to termination are not well-understood. This project will focus on the role of Pol II in the coupling of polyadenylation and termination by characterizing mutated variants of the first and second largest subunits of yeast Pol II that were isolated in a screen for termination defects. This set of mutations identified novel clusters of important residues on the surface of the enzyme that are likely to be interaction sites for other proteins. One specific aim of the project is identification of the protein partners that bind these sites. Standard methods including a yeast two-hybrid approach and a suppressor screen will be used to address this aim. Other mutations altered residues that closely approach the nucleic acids in the elongation complex and so are more likely to affect intrinsic Pol II properties important for the response to termination and/or polyadenylation signals. A second specific aim is to investigate the mechanisms underlying the termination defects for each class of mutants, using a combination of biochemical assays in vitro and functional tests in vivo. Together, results from these experiments will provide a more detailed understanding of the mechanism underlying the drastic changes in the elongation properties of Pol II that occur in response to 3' end processing events.RNA polymerases transcribe the genetic information stored in DNA into messenger RNA, which is then used as a template for protein synthesis. These enzymes are complex machines that must recognize the encoded signals that flank each gene and specify where transcription should start and stop. This research will provide insight into the mechanism by which an RNA polymerase from a model organism uses encoded information to finish and release the RNA and then fall off the DNA for recycling to another gene. Providing resources and projects suitable for training undergraduates was an important consideration in the design of the experiments. Although some of the experiments would be better suited for graduate students, there are other aspects of the project that are particularly appropriate for the more limited experience and the time constraints of undergraduates. The Principal Investigator's research group has a strong track record of mentoring undergraduates, many of whom have gone on to future scientific contributions and careers in academia and industry.