Crystallographic Study of Mechanism of Ribonuclease Ti-Catalysed Specific RNA Hydrolysis

Abstract

Ribonuclease T₁ (RNase T₁) cleaves the phosphodiester bond of RNA specifically at the 2′-end of guanosine. 2′-guanosinemonophosphate (2′-GMP) acts as inhibitor for this reaction and was cocrystallized with RNase T₁. X-Ray analysis provided insight in the geometry of the active site and in the parts of the enzyme involved in the recognition of guanosine. RNase T₁ is globular in shape and consists of a 4.5 turns α-helix lying “below” a four-stranded antiparallel β-sheet containing recognition center as well as active site. The latter is indicated by the position of phosphate and sugar residues of 2′-GMP and shows that Glu58, His92 and Arg77 are active in phosphodiester hydrolysis. Guanine is recognized by a stretch of protein from Tyr42 to Tyr45. Residues involved in recognition are peptide NH and C=O, guanine O₆ and N₁H which form hydrogen bonds and a stacking interaction of Tyr45 on guanine. Although, on a theoretical basis, many specific amino acid-guanine interactions are possible, none is employed in the RNase T₁.guanine recognition.