Document Detail

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.
MedLine Citation:
PMID:  7489519     Owner:  NLM     Status:  MEDLINE    
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.
T B Campbell; T R Cech
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  RNA (New York, N.Y.)     Volume:  1     ISSN:  1355-8382     ISO Abbreviation:  RNA     Publication Date:  1995 Aug 
Date Detail:
Created Date:  1996-01-04     Completed Date:  1996-01-04     Revised Date:  2008-11-20    
Medline Journal Info:
Nlm Unique ID:  9509184     Medline TA:  RNA     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  598-609     Citation Subset:  IM; X    
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
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MeSH Terms
Base Sequence
Binding Sites
DNA Primers
Molecular Sequence Data
RNA / metabolism*
RNA, Catalytic / metabolism*
RNA, Protozoan / metabolism*
Substrate Specificity
Grant Support
Reg. No./Substance:
0/DNA Primers; 0/RNA, Catalytic; 0/RNA, Protozoan; 63231-63-0/RNA

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