Functional Studies of the CDK-module of the Human Mediator Complex

智力上的优点。现代生物学中的一个主要挑战是阐明细胞以组织、信号和发育特有的方式调节数千个基因表达的分子机制。对基因活性的适当调控是每一个主要生物过程的关键要素。专门的蛋白质机器可以以严格调控的方式开启和关闭基因。其中一种被称为Mediator的蛋白质复合体被认为是从酵母到人类的基因活性的关键调节因子，但人们对其作用机制知之甚少。这项研究项目研究了一种被称为CDK模块的介体的四蛋白亚复合体，它可以激活或抑制基因。这个项目的目标是阐明CDK模块激活基因的分子机制，包括它作用的调控步骤，定义催化活性亚基的不同结构域的要求，并描述其他亚基的作用。在确定哺乳动物细胞中明确受CDK-模块调控的特定基因的基础上，先进的生化和分子生物学技术将被用来操纵这种复合体在细胞内的活动，从而获得急需的机械信息。这一研究项目将极大地提高我们对Mediator的理解，以及对基因表达调控机制的总体进化的了解。鉴于介体在基因表达控制中的准普遍作用，该项目的发现将在细胞和分子生物学领域产生深远的影响。此外，以前没有用来研究脊椎动物细胞介体的创新实验方法几乎肯定会提供新的见解。更广泛的影响。该项目的活动将由一个教研小组进行，该小组大力促进科学教育和科学多样性。在实验室工作的本科生和研究生的伙伴关系已取得初步结果，总体男女比例为1：1，实验室成员中有1/3代表少数民族。这笔助学金将资助一个这样的本科生和研究生的合作。主办机构和首席研究人员都积极参加由NSF和其他机构资助的项目，这些项目旨在：a)增加获得博士学位的少数族裔学生的数量，b)为本科生提供实践研究经验，c)为本科生攻读生物科学博士学位做准备，以及d)增加女性在科学和工程领域领导职位的代表性。这些努力的成功体现在以下事实上：这个研究小组的每一份科学出版物都有一名或多名本科生作者，过去的几名本科生现在都在攻读博士学位。首席研究员埃斯皮诺萨博士是当地拉美裔社区的活跃成员，也是加州大学博尔德分校公认的科学教育家，在那里，他是一门约150名学生的本科生课程的讲师，也是他所在部门研究生项目的联合讲师。他在这两个地点教授与他的研究相关的主题。这笔赠款将有助于在这一新兴群体中巩固综合研究和教育的文化，并促进东道国机构的多样性。

Intellectual merit. A major challenge in modern biology is to elucidate the molecular mechanisms employed by cells to regulate expression of thousands of genes in a tissue-, signaling-, and development-specific manner. Proper regulation of gene activity is a key element of every major biological process. Specialized protein machineries can turn genes ON and OFF in a tightly regulated manner. One such protein complex, known as Mediator, is recognized as a critical regulator of gene activity from yeast to humans, yet little is known about its mechanism of action. This research project investigates a four-protein sub-complex of Mediator known as the CDK-module, which can function to either activate or repress genes. The goal of this project is to elucidate the molecular mechanism by which the CDK module activates genes including the regulatory steps at which it acts, defining the requirement for different domains of the catalytically active subunit, and delineating the roles of the other subunits. Building on the identification of specific genes in mammalian cells that are clearly regulated by the CDK-module, sophisticated biochemical and molecular biology techniques will be employed to manipulate the activity of this complex inside cells and thus obtain much needed mechanistic information. This research project will greatly improve our understanding of Mediator in particular and the evolution of gene expression control mechanisms in general. Given the quasi-universal role of Mediator in the control of gene expression, the discoveries arising from this project will have far reaching implications in the fields of cell and molecular biology. Moreover, the innovative experimental approaches not previously utilized to study Mediator in vertebrate cells will almost certainly provide novel insights. Broader impacts. The activities in this project will be carried out by a teaching-research team that strongly promotes science education and diversity in science. Preliminary results have been obtained by partnerships of undergraduate and graduate students working in a laboratory with an overall 1:1 female to male ratio and 1/3 of the lab members representing ethnic minorities. This grant will fund one such undergraduate-graduate student collaboration. Both the host institution and the Principal Investigator actively participate in programs funded by NSF and other agencies that aim to: a) increase the number of minority students earning PhDs, b) provide hands-on research experience to undergrads, c) prepare undergraduate students to pursue PhDs in the biosciences, and d) increase the representation of women in leadership positions in science and engineering. The success of these endeavors is demonstrated by the fact that every scientific publication from this research group has one or more undergraduate authors, and that several of the past undergraduate trainees are now enrolled in PhD programs. The Principal Investigator, Dr. Espinosa, is an active member of the local Hispanic community and a recognized science educator at CU-Boulder, where he is the instructor of a ~150-student undergraduate course and co-instructor in the graduate program of his home Department. He teaches topics related to his research at both venues. This grant will help to cement a culture of integrated research and education within this burgeoning group, as well as to promote diversity at the host institution.