The hairpin ribozyme catalyzes RNA cleavage in
partially hydrated RNA films in the absence of added divalent
cations. This reaction exhibits the characteristics associated
with the RNA cleavage reaction observed under standard
conditions in solution. Catalysis is a site-specific intramolecular
transesterification reaction, requires the 2′-hydroxyl
group of substrate nucleotide A−1, and
generates 2′,3′-cyclic phosphate and 5′-hydroxyl
termini. Mutations in both ribozyme and substrate abolish
catalysis in hydrated films. The reaction is accelerated
by cations that may enhance binding, conformational stability,
and catalytic activity, and is inhibited by Tb3+.
The reaction has an apparent temperature optimum of 4 °C.
At this temperature, cleavage is slow (kobs:
2 d−1) and progressive, with accumulation
of cleavage products to an extent of 40%. The use of synthetic
RNAs, chelators, and analysis of all reaction components
by inductively coupled plasma-optical spectrophotometry
(ICPOES) effectively rules out the possibility of contaminating
divalent metals in the reactions. Catalysis is minimal
under conditions of extreme dehydration, indicating that
the reaction requires hydration of RNA by atmospheric water.
Our results provide a further caution for those studying
the biochemical activity of ribozymes in vitro and in cells,
as unanticipated catalysis could occur during RNA manipulation
and lead to misinterpretation of data.