  Ions interacting with hydrated RNA play a central role in defining its
secondary and tertiary structure. While spatial arrangements of ions, water
molecules, and phosphate groups have been inferred from X-ray studies, the role
of electrostatic and other noncovalent interactions in stabilizing compact
folded RNA structures is not fully understood at the molecular level. Here, we
demonstrate that contact ion pairs of magnesium (Mg2+) and phosphate groups
embedded in local water shells stabilize the tertiary equilibrium structure of
transfer RNA (tRNA). Employing dialyzed tRNAPhe from yeast and tRNA from
Escherichia coli, we follow the population of Mg2+ sites close to phosphate
groups of the ribose-phosphodiester backbone step by step, combining linear and
nonlinear infrared spectroscopy of phosphate vibrations with molecular dynamics
simulations and ab initio vibrational frequency calculations. The formation of
up to six Mg2+/phosphate contact pairs per tRNA and local field-induced
reorientations of water molecules balance the phosphate-phosphate repulsion in
nonhelical parts of tRNA, thus stabilizing the folded structure
electrostatically. Such geometries display limited sub-picosecond fluctuations
in the arrangement of water molecules and ion residence times longer than 1
{\mu}s. At higher Mg2+ excess, the number of contact ion pairs per tRNA
saturates around 6 and weakly interacting ions prevail. Our results suggest a
predominance of contact ion pairs over long-range coupling of the ion
atmosphere and the biomolecule in defining and stabilizing the tertiary
structure of tRNA.
