Sp. Ryder et Sa. Strobel, Nucleotide analog interference mapping of the hairpin ribozyme: Implications for secondary and tertiary structure formation, J MOL BIOL, 291(2), 1999, pp. 295-311
The hairpin ribozyme is a small, naturally occurring RNA capable of folding
into a distinct three-dimensional structure and catalyzing a specific phos
phodiester transfer reaction. We have adapted a high throughput screening p
rocedure entitled nucleotide analog interference mapping (NAIM) to identify
functional groups important for proper folding and catalysis of this riboz
yme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to
determine the contribution made by individual ribose 2'-OH and purine funct
ional groups to the hairpin ribozyme ligation reaction. Substitution with 2
'-deoxy-nucleotide analogs disrupted activity at six sites within the riboz
yme, and a unique interference pattern was observed at each of the 11 conse
rved purine nucleotides. Ln most cases where such information is available,
the NAIM data agree with the previously reported single-site substitution
results. The interference patterns are interpreted in comparison to the iso
lated loop A and loop B NMR structures and a model of the intact ribozyme.
These data provide biochemical evidence in support of many, but not all, of
the noncanonical base-pairs observed by NMR in each loop, and identify the
functional groups most likely to participate in the tertiary interface bet
ween loop A and loop B. These groups include the 2'-OH groups of A10, G11,
U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of
A9 and A24. The data also predict non-A form sugar pucker geometry at U39
and U41. Based upon these results, a revised model for the loop A tertiary
interaction with loop B is proposed. This work defines the chemical basis o
f purine nucleotide conservation in the hairpin ribozyme, and provides a ba
sis for the design and interpretation of interference suppression experimen
ts. (C) 1999 Academic Press.