       Document 0713
 DOCN  M9630713
 TI    Identification of ribozymes within a ribozyme library that efficiently
       cleave a long substrate RNA.
 DT    9603
 AU    Campbell TB; Cech TR; Howard Hughes Medical Institute, Department of
       Chemistry and; Biochemistry, University of Colorado, Boulder 80309-0215,
       USA.
 SO    RNA. 1995 Aug;1(6):598-609. Unique Identifier : AIDSLINE MED/96079988
 AB    Positions 2-6 of the substrate-binding internal guide sequence (IGS) of
       the L-21 Sca I form of the Tetrahymena thermophila intron were
       mutagenized to produce a GN5 IGS library. Ribozymes within the GN5
       library capable of efficient cleavage of an 818-nt human
       immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were
       identified by ribozyme-catalyzed guanosine addition to the 3' cleavage
       product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the
       GN5 library that actively cleaved the long substrate were characterized
       kinetically and compared to the wild-type ribozyme (GGAGGG) and two
       control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have
       specific sites within the long substrate, but were not identified during
       screening of the library. Under single-turnover conditions, ribozymes
       GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold
       faster than control ribozymes. Short cognate substrates, which should be
       structureless and therefore accessible to ribozyme binding, were cleaved
       at similar rates by all ribozymes except GGGGCU, which showed a fourfold
       rate enhancement. The rate of cleavage of long relative to short
       substrate under single-turnover conditions suggests that GGCUCC and
       GUGGCU were identified because of accessibility to their specific
       cleavage sites within the long substrate (substrate-specific effects),
       whereas GGGGCU was identified because of an enhanced rate of substrate
       binding despite a less accessible site in the long substrate. Even
       though screening was performed with 100-fold excess substrate (relative
       to total ribozyme), the rate of multiple-turnover catalysis did not
       contribute to identification of trans-cleaving ribozymes in the GN5
       library.
 DE    Animal  Base Sequence  Binding Sites  DNA Primers  Molecular Sequence
       Data  RNA/*METABOLISM  RNA, Catalytic/*METABOLISM  RNA,
       Protozoan/*METABOLISM  Substrate Specificity  Support, U.S. Gov't,
       P.H.S.  Tetrahymena  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

