Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity.
The RNA substrate-binding site of the Tetrahymena ribozyme is connected to the catalytic core by the joining region J1/2. Although J1/2 is not conserved among group I introns, small insertions or deletions in this sequence have dramatic effects, enhancing the turnover number and sequence specificity of ribozyme-catalyzed RNA cleavage. Measurements of rate constants for individual steps in the reaction have revealed the basis of these improvements. Ironically, the higher turnover and specificity both result from decreased affinity for RNA, rather than better cleavage. These results provide evidence that the nonconserved J1/2 sequence positions the RNA substrate to optimize tertiary interactions and ensure cleavage at the position corresponding to the 5' splice site. The wild-type RNA is well adapted to its biological function, and its limitations in multiple turnover can be corrected by mutation.
Young
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Department of Molecular, Cellular and Developmental Biology, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215.
Herschlag
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D
Cech
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TR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology / Q000235:genetics*; D013816:Thermodynamics
D016337:RNA, Catalytic