Selection of the 3' end mRNA processing site by Cleavage Factor I

智力优点：基因表达是利用作为DNA储存在细胞核中的信息合成功能蛋白质的过程，并以mRNA分子的形式传递到蛋白质合成的细胞位点，通过复制（转录）DNA产生。基因表达的一个重要步骤是通过识别特定RNA序列的3 '端加工酶精确修剪信使RNA（mRNA）分子的3'端。这个过程决定了每个mRNA分子的长度，并影响其作为蛋白质合成模板的效率。大约有一半的人类基因含有一个以上的3 '端加工的潜在位点，并受到替代加工。 选择合适的加工位点在参与免疫应答和细胞分化的蛋白质合成中特别重要。 该项目使用酵母作为模型系统来研究3 '-末端加工的机制，并专注于一种称为切割因子I（CF I）的五种蛋白质的进化上保守的复合物，该复合物识别定义3'-末端加工位点的mRNA信号序列。 该研究将利用单粒子电子显微镜，结合RNA足迹和分子建模技术来构建CF I整体结构的模型，并阐明它如何识别mRNA信号序列并指导mRNA 3 '端的加工。 酵母遗传学，加上生化结合实验，将用于验证和完善所产生的分子模型。 这项工作具有根本的重要性，因为mRNA 3 '端加工影响所有真核细胞中所有蛋白质的表达。 更广泛的影响：该项目将为塔夫茨生物化学项目的研究生，博士后研究员和暑期实习生提供实践研究培训，包括将通过NIH赞助的题为“在生物医学科学中建立多样性”的项目招募的本科生和高中生，他们将通过塔夫茨少数民族事务办公室管理的教师和高中生计划来到实验室。Bohm继续他在塔夫茨生物化学计划和塔夫茨PREP计划中的导师活动，该计划是一个学士后计划，为来自代表性不足的群体的学生准备研究生院。此外，这项工作中使用的技术将被整合到博姆博士目前教授的两门课程中。 第一个是分子识别的研究生课程。 第二个是夏季研讨会式的教室/实验室课程，旨在介绍参与者在自己的研究中使用散射方法来分析大分子复合物。 因此，这门课程是传播研究技术方面的一个特别重要的机制，并帮助其他人将这些方法应用于其他生物系统。

Intellectual merit: 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. Broader impacts: 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.