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

智力优势：基因表达是利用存储在细胞核中的信息作为DNA合成功能蛋白质的过程，并以复制(转录)DNA产生的mRNA分子的形式传输到蛋白质合成的细胞部位。基因表达的一个重要步骤是通过识别特定RNA序列的3‘端加工酶精确地修剪信使RNA(MRNA)分子的3’端。这一过程决定了每个信使核糖核酸分子的长度，并影响其作为蛋白质合成模板的效率。大约一半的人类基因包含一个以上的潜在3‘端加工位点，并受到替代加工的影响。在参与免疫反应和细胞分化的蛋白质的合成过程中，选择合适的加工位点尤为重要。本项目以酵母为模型系统来研究3‘端加工的机制，重点研究了一种进化上保守的由五种蛋白质组成的复合体，称为切割因子I(CFI)，它识别定义3’端加工位点的mRNA信号序列。这项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CFI的整体结构模型，并阐明它是如何识别mRNA信号序列和指导mRNAs 3‘端的加工的。酵母遗传学，结合生化结合实验，将被用来验证和改进所产生的分子模型。这项工作具有根本的重要性，因为mRNA3‘端处理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赞助的名为“在生物医学科学中建立多样性”的计划招收的本科生，以及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的高中生。这笔资金还将允许博姆博士在塔夫茨生物化学项目和塔夫茨预科项目中继续他的导师活动，塔夫茨预科项目是一个学士学位后项目，为来自代表性不足群体的学生进入研究生院做准备。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。第一个是关于分子识别的研究生课程。第二个是暑期工作坊风格的课堂/实验室课程，旨在向参与者介绍在他们自己的研究中使用散射方法来分析大分子络合物。因此，这门课程是传播研究的技术方面并帮助其他人将这些方法应用于其他生物系统的一个特别重要的机制。

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.