Study of temperature-sensitive mutants of yeast guanylate kinase
EMSL Project ID
37696
Abstract
The ability to switch genes on and off spatially and temporally is important for understanding individual gene function in complex biological processes. Despite the fact that a variety of conditional control systems have been developed that act through modifying DNA, RNA, or proteins, none of them is universally applicable to all organisms. There is a clear need to develop more general, precise, and adaptable systems. Temperature-sensitive (Ts) mutants are a class of conditional alleles that exhibit wild-type activity at a permissive temperature but diminished activity at a restrictive temperature. Ts mutants have great potential to be developed into a precise and easy-to-use conditional control system, and have been used in many organisms to investigate gene function in vivo, including viruses, bacteria, S. cerevisiae (yeast), Drosophila, C. elegans, and mammalian cell cultures. However, the use of Ts alleles is hampered by the standard experimental approach of forward genetic screening that is time-consuming and not feasible for organisms for which screening procedures do not exist. Rational design and construction of Ts alleles is an attractive alternative approach. This requires computational prediction of amino acid substitutions likely to confer a Ts phenotype, followed by construction of highly ranked candidate mutants via site-directed mutagenesis. We have developed a computational method, TSPRED, which evaluates and ranks all possible single amino acid substitutions in a protein according to the probability of being a Ts mutation. We validated predicted Ts mutations via site-directed mutagenesis for three yeast essential genes: histone acetyltransferase ESA1, guanylate kinase GUK1, and pantothenate kinase CAB1. The main goal of this proposal is to characterize molecular mechanisms of Ts proteins by NMR technology. Using yeast Guk1 as a model system, we propose to obtain the temperature dependence of NMR chemical shifts combined with three-dimensional structural information of Ts proteins. Since we use 25°C as the permissive temperature and 37°C as the restrictive temperature, NMR technology is perfectly suited for the characterizing Ts proteins as compared to crystallization. Results from the proposed work can feedback to our computational framework for predicting ideal Ts mutants that will allow effectively characterize new gene functions broadly applicable in model systems from bacteria to mammalian cell culture.
Project Details
Project type
Limited Scope
Start Date
2009-09-23
End Date
2009-11-23
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members