| Co-Investigator(Kenkyū-buntansha) |
OHTSUKA Eiko Faculty of Pharmaceutical Sciences, Hokkaido University, 薬学部, 教授 (80028836)
KYOGOKU Toshimasa Protein Research Institute, Osaka University, 蛋白質研究所, 教授 (90012632)
TOMITA Ken-ichi Faculty of Pharmaceutical Sciences, Osaka University, 薬学部, 教授 (30028831)
UESUGI Seiichi Faculty of Pharmaceutical Sciences, Osaka University, 薬学部, 助教授 (70028851)
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| Research Abstract |
The object of this research project is to elucidate recognition mechanism between proteins and nucleic acids. The target proteins are mainly RNase Tl, Which recognizes a guanine base of RNA and hydrolyzes the phosphodiester bond, and a regulation protein <lambda> cro) which binds to an operator DNA <lambda> O_r3) and controls the gene expression.
1. X-Ray Crystallographic study. (1) Structures of RNase T1-2'-GMP (1.9 <ang> resolution) and RNase T1-3'GMP (2.6 A resolution) complexes were determined. The substrate recognition site was assigned in detail for the first time. (2) A complex between RNase Tl and a substrate analogue, dGflpU, was crystallized. (3) A complex between an RNase Tl mutant and 2'GMP was crystallized. The crystal structures of these complexes are now under investigation.
2. Synthesis of an RNase Tl its mutant genes and their expression to study the structure-activity relationships. (1) An RNase Tl gene was prepared from chemically synthesized deoxyoligonucleotide fragm
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ents. A system for its expression in E. coli was constructed. (2) More than 20 RNase Tl mutants, which are designed taking the results of X-ray crystallographic studies into consideration, were prepared. The structure-activity relationships of RNase Tl were analyzed. The results substanciated stacking interactions with Tyr 42 and Tyr 45 and hydrogen bonding interactions with Asn 43, Asn 44 and Glu 46 are important for recognition of the substarte. A new mechanism for RNase Tl catalysis, which assumes a direct involvement of His 40 and His 92 in constrast to the previous one, was proposed.
3. Interactions between operator DNA's and cro proteins. (1) Operator DNA's <lambda> O_r3 17mer and <phi>80 O_r2 19mer, and CRP binding site DNA, 22mer were chemically synthesized. (2) A system for efficient production of the cro protein in E. coli was constructed using a recA promoter. The amino acid residues exposed on the surface were assigned by NMR. NMR study also revealed that dissociation of the dimeric form and denaturation occur at 50゜C. (3) NMR study on the cro-operator complex showed that the O_r3 DNA bends at the center of the helix and the dimeric structure structure of cro undergoes a big change upon association. Less
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