BINDING OF GUANOSINE AND 3' SPLICE SITE ANALOGS TO A GROUP-I RIBOZYME- INTERACTIONS WITH FUNCTIONAL-GROUPS OF GUANOSINE AND WITH ADDITIONAL NUCLEOTIDES
S. Moran et al., BINDING OF GUANOSINE AND 3' SPLICE SITE ANALOGS TO A GROUP-I RIBOZYME- INTERACTIONS WITH FUNCTIONAL-GROUPS OF GUANOSINE AND WITH ADDITIONAL NUCLEOTIDES, Biochemistry, 32(19), 1993, pp. 5247-5256
Dissociation constants, K(d), were measured by equilibrium dialysis at
5-degrees-C for a series of substrates binding to the L-21 ScaI riboz
yme derived from the Tetrahymena thermophila self-splicing large subun
it (LSU) ribosomal RNA intron. These substrates are analogues for the
3' exon splice site, the cyclization site, and the exogenous G that in
itiates group I splicing. UCG has a K(d) of 17 muM. Lengthening the su
bstrate to GUCG and GGUCG enhances binding but by less than expected f
rom potential base pairing. Functional groups on the 3'-terminal G of
GUCG were replaced with H to test their effect on binding. GUC(2'dG) b
inds slightly tighter than the all-ribose molecule but shows no reacti
vity as a substrate. GUC(3'dG) binds weaker than GUCG. Inosine and 2-a
minopurine ribonucleoside at the 3' position weaken binding by 16- and
26-fold, respectively, but both tetramers are reactive. Thus hydrogen
bonds to Watson-Crick pairing positions of the 3'G of GUCG contribute
1-2 kcal/mol to the free energy change for binding. Similar results a
re found in comparisons of UCG with UC(2'dG), UC(3'dG), and UCI. The n
onreactive substrate GUCdGA includes a phosphodiester bond 3' to the g
uanosine that is the site of chemistry for the all-ribose substrate GU
CGA; GUCdGA binds 50 times more weakly than GUCdG. A similar result is
obtained for GUCdGU. Competition experiments show that guanosine and
guanosine 5'-monophosphate bind with dissociation constants of about 0
.9 mM. The monomers 2'dG and 3'dG have K(d)'s of 0.5 and greater-than-
or-equal-to 3 mM, respectively. This suggests that sugar pucker and/or
interactions with hydroxyl groups affect binding. Implications for ri
bozyme catalysis, splicing, cyclization, and design of antisense oligo
mers are discussed.