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.
