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Nucleic Acids Research, 1991, Vol. 19, No. 14 3875-3880
© 1991

MOLECULAR BIOLOGY

A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3' end blocked for transesterification

Cheryl A. Grosshans and Thomas R. Cech*

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 80309-0215, USA

* To whom correspondence should be addressed

Received May 10, 1991. Revised June 17, 1991. Accepted June 17, 1991.

The L-21 Seal form of the Tetrahymena ribozyme acts as a sequence-specific endonuclease. This ribozyme has a homogeneous 5' end but a somewhat heterogeneous 3' end, as is typical of RNA synthesized by transcription in vitro. To produce a more homogeneous ribozyme for both structural and enzymological studies, a hammerhead ribozyme was inserted at the 3' end of the Tetrahymena ribozyme. During transcription the hammerhead moiety self-cleaves to produce the L-21 A Tetrahymena ribozyme, which ends at A410 with a 2',3'-cyclic phosphate terminus. The new ribozyme has endoribonuclease activity equivalent to that of L-21 Seal under conditions where binding of substrate is rate-limiting, as well as under conditions where chemical cleavage by guanosine is rate-limiting. However, the L-21 A has lost activity in oligo(C) dlsproportionation (e.g., 2 pC5-pC4 + pC6), consistent with the previous proposal that this reaction occurs predominantly through a covalent ribozyme-substrate intermediate involving the 3'-terminal hydroxyl group of the ribozyme. Formation of such an intermediate would be prevented by the 2',3'-cyclic phosphate terminus. Thus the L-21 A ribozyme has simplified enzymatic activity, being fully active as an endonuclease but blocked for disproportionation.


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