Molecular Dissection of Transcription Termination Mechanisms in Eukaryotes

Tufts University, Medford MA

Investigators

Abstract

Intellectual Merit. The transcription of eukaryotic mRNA is an obligate step in the flow of information from the genome to expression of proteins needed by the cell. The final step of transcription is called termination, and involves release of RNA polymerase II (Pol II) and the RNA from the DNA template. Defects in termination can impair cell function due to the interference of read-through transcription on downstream DNA elements needed for DNA replication, chromosomal segregation, or the initiation of transcription. Poor termination can also lead to decreased processing and increased degradation of the RNA as well as reduced initiation at the gene's promoter. Despite significant advances in recent years, termination remains one of the least understood steps of transcription. Pol II termination downstream of mRNA poly(A) sites requires the concerted efforts of the Rat1 exonuclease and proteins which recognize and act to cleave RNA at the poly(A) site. Transcription by Pol II through a gene's body is both rapid and processive, yet if stalled, the association of Pol II with DNA is remarkably stable. Nevertheless, interactions of termination factors with the polymerase overcome these challenges and induce changes that lead to pausing and release. The goal of this research is to decipher the molecular mechanisms that lead to Rat1-mediated termination of Pol II and to seek parallels in how Rat1 also facilitates release of Pol I, which transcribes ribosomal RNA. Through genetic screens in yeast, using both directed and random mutagenesis, this project should identify critical regions of RNA polymerase that might interact directly with termination factors or otherwise alter the termination behavior of the enzyme. Previously, the lack of a defined, easily manipulated in vitro system in which to study termination has made it difficult to determine individual contributions of the various factors. This problem has been addressed by developing a new in vitro assay in which mutant and wild-type polymerase can be stalled and then challenged with purified factors alone and in combination. This assay will be incorporated into an integrated strategy that examines transcription in vivo and in cell extract. Broader impacts. Scientifically, the successful completion of this project should give significant new insight into the mechanism of transcription termination and lead to a new fundamental understanding of points at which this step in the transcription cycle might be regulated in all eukaryotes. Furthermore, it will provide rigorous training for students in the disciplines of biochemistry, molecular biology and genetics as well as multiple opportunities for trainees to gain experience in the written and oral presentation of their research and in mentoring younger students. In addition, the PI has been active in developing new ways to train the next generation of scientists and to increase diversity in biological research, and the members of the PI's lab participate in these programs as mentors and trainees. These programs include summer research and postbaccalaureate programs for underrepresented students, and a postdoctoral training program that prepares fellows for successful academic careers that involve research, mentoring and teaching undergraduates in the biological sciences.