Analysis of Termination Mutants of RNA Polymerase II

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

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.