STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

环磷酸腺苷受体蛋白(CRP)是一种调节因子。 在大肠杆菌中转录20多个基因。CAMP结果的约束力 在CRP所需的构象变化中。MBRS项目涉及 C反应蛋白Gly-200突变为Phe。这种甘氨酸提供了所需的转折力 对于相邻的Beta-11和Beta-12的适当构象 思特斯。该突变会将GGTAAA(Gly-Lys)转换为TTTAAA(Phe-Lys) 提供大量氨基酸残基以干扰构象 参与了显然是必要的Beta Sheet结构 CRP的C-末端结构域。两个底座的变化也形成了一个圆锥体 监察大桥成功建造的限制地点 迷你报中所需的突变。这很有用，因为目前还不清楚 突变将导致CRP活性的变化，这将允许 用平板法进行表型选择。这个项目是合理的。 并将为MBR学者提供一系列 实验经验在当今分子生物学中很重要。这些 包括噬菌体生产和分离所需的程序 单链DNA、定点突变和双脱氧DNA C反应蛋白突变体的测序、转化、筛选、Beta检测 半乳糖苷酶活性、突变株CRPS的纯化至均一 以及几种用于表征该细胞的体外实验 C反应蛋白的结构/功能特性。 学生将在实验室开始他/她的任期，通过协助和 向其中一名研究生学习技术。在某个时候 在他们的学徒期(在我评估他们的进步时)，他们将开始 更独立于他们的直接导师的工作。我讨论的是 结果，提出建议，协助解释数据，批评 在适当的情况下。学生将一起参加实验室讨论 和我的其他学生在一起，可能会参加ASBCMB会议，预计 在MBRS全国会议上展示这项研究。

The cyclic AMP receptor protein (CRP) acts as a regulatory factor for the transcription of more than 20 genes in E. coli. Binding of cAMP results in a required conformational change in CRP. The MBRS project involves mutation of CRP Gly-200 to Phe. This glycine provides the turn required for the appropriate conformation of the adjacent Beta-11 and Beta-12 strands. The mutation will convert GGTAAA (Gly-Lys) to TTTAAA (Phe-Lys) providing a bulky amino acid residue to interfere with the conformation involved in what is apparently an essential Beta sheet structure in the C-terminal domain of CRP. The two bases change also forms a Dral restriction site for monitoring the successful construction of the desired mutation in minipress. This is useful since it is not clear that the mutation will cause a change in CRP activity which would allow for phenotypic selection by a plate assay. The project is reasonable straight forward and will provide the MBRS scholar with a series of experimental experiences important in current molecular biology. These include the procedures required for phagemid production and isolation of the single stranded DNA, site specific mutagenesis and dideoxy DNA sequencing, transformation, selection of CRP mutants, assays for Beta galactosidase activity, purification of the mutant CRPs to homogeneity and the several in vitro assays used to characterize the structure/function properties of CRP. The student will begin his/her tenure in the laboratory by assisting and learning techniques from one of the graduate students. At some time during their apprenticeship (as I assess their progress) they will begin to work more independently of their immediate mentor. I discuss the results, make suggestions, assist in interpreting the data, criticize where appropriate. The student will participate in lab discussions along with my other students and may attend an ASBCMB Meeting and are expected present the research at an MBRS national meeting.