title: Domain swapping in ribonuclease T1 allows the acquisition of double-stranded activity.
abstract: A mutant of ribonuclease T1 (RNase T1), denoted RNase Talpha, that is designed to recognize double-stranded ribonucleic acid was created. RNase Talpha carries the structure of RNase T1 except for a part of its loop L3 domain, which has been swapped for a corresponding domain from alpha-sarcin. The RNase Talpha maintains the pleated beta-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase Talpha-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced K(m) value of 6.94 x 10(-7) M. When the stranded specificity is examined, RNase Talpha catalyzes the hydrolysis of guanine base not only of single-stranded but also, as by design, of double-stranded RNA. The change of stranded specificity suggests the feasibility of using domain swapping to make a substrate-specific ribonuclease. This study suggests that the loop L3 in RNase T1 can be used as a 'cassette player' for inserting a functional domain to make ribonuclease of various specificities.
authors.affiliation: Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei, Taiwan.
authors.forename: Dow-Tien
authors.initials: DT
authors.lastname: Chen
authors.forename: Alan
authors.initials: A
authors.lastname: Lin
mesh_terms: D001483:Base Sequence; D006147:Guanine / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D006147:Guanine; D006163:Ribonuclease T1