DNase I–tRNA Interaction

Abstract

ABSTRACT

Deoxyribonuclease I (DNase I) binds right-handed DNA duplex via a minor groove and the backbone phosphate group with no contact to the major groove. It hydrolyses double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions, in the presence of divalent Mg and Ca cations. Even though DNase–RNA interaction was observed, less is known about the protein–RNA binding mode and the effect of such complexation on both protein and RNA conformations. The aim of this study was to examine the effects of DNase I–tRNA interaction on tRNA and protein conformations. The interaction of DNase I with tRNA is monitored under physiological conditions, in the absence of Mg²⁺, using constant DNA concentration of 12.5 mM (phosphate) and various protein contents (10 μM to 250 μM). FTIR, UV-visible, and CD spectroscopic methods were used to analyze the protein binding mode, the binding constant, and the effects of polynucleotide–enzyme interaction on both tRNA and protein conformations. Spectroscopic evidence showed major DNase–PO₂ and minor groove interactions with overall binding constant of K = 2.1 (±0.7) × 10⁴ M⁻¹. The DNase I–tRNA interaction alters protein secondary structure with major reduction of the α-helix, and increases the random coil, β-anti and turn structures, while tRNA remains in the A-conformation. No digestion of tRNA by DNase I was observed in the protein–tRNA complexes.