A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3' end blocked for transesterification.
The L-21 Scal 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 Scal 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) disproportionation (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.
Grosshans
C A
CA
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Cech
T R
TR
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D004951:Esterification; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000096:biosynthesis / Q000235:genetics; D013768:Tetrahymena / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D016053:RNA, Protozoan; D004720:Endonucleases; C047020:endodeoxyribonuclease ScaI; D015252:Deoxyribonucleases, Type II Site-Specific