Gene expression is the process of synthesizing functional proteins using information stored in the nucleus as DNA and transmitted to cellular sites of protein synthesis in the form of mRNA molecules, produced by copying (transcribing) DNA. An essential step in gene expression is the precise trimming of the 3'-ends of messenger RNA (mRNA) molecules by 3'-end processing enzymes that recognize specific RNA sequences. This process determines the length of each mRNA molecule and affects the efficiency with which it is utilized as a template for protein synthesis. Roughly half of human genes contain more than one potential site of 3'-end processing and are subject to alternative processing. Selection of the appropriate processing site is particularly important in the synthesis of proteins involved in immune responses and in cell differentiation. This project uses yeast as a model system to study the mechanism of 3'-end processing and focuses on an evolutionarily conserved complex of five proteins called Cleavage Factor I (CF I) that recognizes mRNA signal sequences which define the sites of 3'-end processing. The research will utilize single-particle electron microscopy, coupled with RNA foot-printing and molecular modeling techniques to construct models of the overall structure of CF I and elucidate how it recognizes mRNA signal sequences and directs processing of the 3'-ends of mRNAs. Yeast genetics, coupled with biochemical binding experiments, will be used to verify and refine the resulting molecular models. This work is of fundamental importance because mRNA 3'-end processing affects the expression of all proteins in all eukaryotic cells.
The project will provide hands-on research training for the graduate students in the Tufts Biochemistry program, postdoctoral fellows and summer trainees, including undergraduates who will be recruited through an NIH-sponsored program entitled "Building Diversity in the Biomedical Sciences" and high school students who will come to the laboratory through the Teachers and High School Students Program administered by the Tufts Office of Minority Affairs. The funds will also permit Dr. Bohm to continue his mentorship activities within the Tufts Biochemistry Program and within the Tufts PREP program, a postbaccalaureate program that prepares students from underrepresented groups for graduate school. In addition, the techniques used in this work will be integrated into two courses Dr. Bohm currently teaches. The first is a graduate course on molecular recognition. The second is a summer workshop-style classroom/laboratory course designed to introduce participants in the use of scattering methods to analyze macromolecular complexes in their own research. Thus, this course is a particularly important mechanism for disseminating the technical aspects of the research and helping others apply these methods to other biological systems.