Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.
RNA. 1995 Aug;1(6):598-609. Unique Identifier : AIDSLINE MED/96079988 Campbell TB; Cech TR; Howard Hughes Medical Institute, Department of Chemistry and; Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Abstract:
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.
Keywords: 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 960330
M9630725
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Important note: Information in this article was accurate in 1996. The state of the art may have changed since the publication date.
