Replacement of the conserved G.U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity, and fidelity

Biochemistry. 1994 Nov 22;33(46):13856-63. doi: 10.1021/bi00250a040.


There is a phylogenetically conserved G.U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 degrees C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G.U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Composition
  • Base Sequence
  • Catalysis
  • Cytosine / metabolism*
  • Guanine / metabolism*
  • Hot Temperature
  • Introns
  • Kinetics
  • Molecular Sequence Data
  • Nucleic Acid Denaturation
  • Oligonucleotides / metabolism
  • RNA Splicing
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism*
  • RNA, Protozoan / chemistry
  • RNA, Protozoan / metabolism
  • Tetrahymena / enzymology*
  • Uridine / metabolism*


  • Oligonucleotides
  • RNA, Catalytic
  • RNA, Protozoan
  • Guanine
  • Cytosine
  • Uridine