Replacement of the conserved G.U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity, and fidelity.
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)
Pyle
A M
AM
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Moran
S
S
Strobel
S A
SA
Chapman
T
T
Turner
D H
DH
Cech
T R
TR
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002384:Catalysis; D003596:Cytosine / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism*; D006358:Hot Temperature; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology*; D014529:Uridine / Q000378:metabolism*
D009841:Oligonucleotides; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D006147:Guanine; D003596:Cytosine; D014529:Uridine