| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1987: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Ribonuclease Tl (RNase Tl) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. Because of its high specificity, RNase Tl has been a useful enzyme for RNA sequencing since its discovery together with RNase A which is specific for cytosine and uridine. RNase Tl is a small, stable globular protein (104 amino acids, Mr 11000), hence it has been studied by many methods such as protein chemistry, enzyme kinetics and physicochemistry (NMR, CD) etc. The tertiary structure of RNase Tl-2'-GMP complex was solved by X-ray crystallography. Following this study, we initiated a study by protein engineering to solve the problem of how RNase Tl recognizes RNA molecule and catalyzes its hydrolysis.
We synthesized the gene for RNase Tl and succeeded in expressing it in E. coli by the fused-protein and secretion methods, and also prepared many mutants by site-directed mutagenesis. The enzyme kinetics of these mutants were analyzed by using pGpC as a su
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bstrate. Mutants which we made were grouped into the following two categories based on their functions.
(1) Mutants at the guanine base recognition residues, Tyr 42, Asn 43, Asn 44, Tyr 45 and Glu 46. The guanine base is assumed to be recognized by RNase Tl through two types of interactions, hydrogen bonding and stacking, at the Tyr 42 to Glu 46 site from the results of X-ray analysis. We were able to characterize the function of side chains of Asn 43, Asn 44 and Glu 46, which interacts with the guanine base by hydrogen-bonding, from the kinetic data. By chainging Tyr 45 to Trp, the stacking effect was increased and this mutant showed a higher nucleolytic activity than that of the native RNase Tl.
(2) Catalytic residues, His 40, Glu 58, Arg 77 and His 92. The roles of catalytic residues, His 40, Glu 58 and His 92, has been speculated from the results obtained mainly from chemical modification experiments. However, we proposed a new reaction mechanism by utilizing the site-directed mutagenesis method. Less
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