Solution Structure of Loop A from the Hairpin Ribozyme from Tobacco Ringspot Virus Satellite

Abstract

The solution structure of loop A from the hairpin ribozyme found in the minus strand of tobacco ringspot virus satellite has been determined by NMR spectroscopy. The ribozyme consists of two internal loops flanked by short helices: loop A and helices I and II include the substrate and substrate binding site; loop B and helices III and IV are the catalytic domain. Loop A is a symmetric internal loop of eight nucleotides that contains the cleavage site. The 2-amino group of the guanine immediately 3' to the cleavage site is essential for catalysis. NMR results show that this guanine forms a sheared G.A base pair. The cytosine residue immediately 5' to the cleavage site forms an AH+.C base pair with an adenine whose pKa is shifted to 6.2 to allow partial protonation near neutral pH. Although the residues flanking the cleavage site are stacked in an A-form pattern, the phosphodiester backbone next to the cleavage site on the 3' side is splayed apart. This places the following base-a uracil-in the expanded major groove. The conformational flexibility and the lack of steric hindrance of the uracil as well as the unoccupied Watson-Crick positions on the sheared G.A base pair can allow loop A to specifically interact with the catalytic domain (loop B) without drastically changing its own conformation. The three-dimensional structure of loop A provides explanations for previously published mutation and structural mapping results.