Optical melting was used to determine the stabilities of 11
small RNA oligomers of defined secondary structure as a function
of magnesium ion concentration. The oligomers included helices
composed of Watson–Crick base pairs, GA tandem base pairs,
GU tandem base pairs, and loop E motifs (both eubacterial and
eukaryotic). The effect of magnesium ion concentration on stability
was interpreted in terms of two simple models. The first assumes
an uptake of metal ion upon duplex formation. The second assumes
nonspecific electrostatic attraction of metal ions to the RNA
oligomer. For all oligomers, except the eubacterial loop E,
the data could best be interpreted as nonspecific binding of
metal ions to the RNAs. The effect of magnesium ions on the
stability of the eubacterial loop E was distinct from that seen
with the other oligomers in two ways. First, the extent of
stabilization by magnesium ions (as measured by either change
in melting temperature or free energy) was three times greater
than that observed for the other helical oligomers. Second,
the presence of magnesium ions produces a doubling of the enthalpy
for the melting transition. These results indicate that magnesium
ion stabilizes the eubacterial loop E sequence by chelating
the RNA specifically. Further, these results on a rather small
system shed light on the large enthalpy changes observed upon
thermal unfolding of large RNAs like group I introns. It is
suggested that parts of those large enthalpy changes observed
in the folding of RNAs may be assigned to variations in the
hydration states and types of coordinating atoms in some
specifically bound magnesium ions and to an increase in the
observed cooperativity of the folding transition due to the
binding of those magnesium ions coupling the two stems together.
Brownian dynamic simulations, carried out to visualize the metal
ion binding sites, reveal rather delocalized ionic densities
in all oligomers, except for the eubacterial loop E, in which
precisely located ion densities were previously calculated.
