RNase A – tRNA binding alters protein conformation

Abstract

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5′ bonds in RNA on the 3′ side of pyrimidine to form cyclic 2′,5′-phosphates. Even though extensive structural information is available on RNase A complexes with mononucleotides and oligonucleotides, the interaction of RNase A with tRNA has not been fully investigated. We report the complexation of tRNA with RNase A in aqueous solution under physiological conditions, using a constant RNA concentration and various amounts of RNase A. Fourier transform infrared, UV-visible, and circular dichroism spectroscopic methods were used to determine the RNase binding mode, binding constant, sequence preference, and biopolymer secondary structural changes in the RNase–tRNA complexes. Spectroscopic results showed 2 major binding sites for RNase A on tRNA, with an overall binding constant of K = 4.0 × 105(mol/L)–1. The 2 binding sites were located at the G-C base pairs and the backbone PO2group. Protein–RNA interaction alters RNase secondary structure, with a major reduction in α helix and β sheets and an increase in the turn and random coil structures, while tRNA remains in the A conformation upon protein interaction. No tRNA digestion was observed upon RNase A complexation.