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Protein Engineering, Vol. 15, No. 12, 997-1003, December 2002
© 2002 Oxford University Press

Domain swapping in ribonuclease T1 allows the acquisition of double-stranded activity

Dow-Tien Chen and Alan Lin1

Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei, Taiwan

A mutant of ribonuclease T1 (RNase T1), denoted RNase T{alpha}, that is designed to recognize double-stranded ribonucleic acid was created. RNase T{alpha} 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 T{alpha} maintains the pleated ß-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase T{alpha}-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced Km value of 6.94x10-7 M. When the stranded specificity is examined, RNase T{alpha} 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.


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