SOLUTION STRUCTURE OF LOOP A FROM THE HAIRPIN RIBOZYME FROM TOBACCO RINGSPOT VIRUS SATELLITE

The solution structure of loop A from the hairpin ribozyme found in th e minus strand of tobacco ringspot virus satellite has been determined by NMR spectroscopy. The ribozyme consists of two internal loops flan ked by short helices: loop A and helices I and II include the substrat e and substrate binding site; loop B and helices III and IV are the ca talytic domain. Loop A is a symmetric internal loop of eight nucleotid es that contains the cleavage site. The 2-amino group of the guanine i mmediately 3' to the cleavage site is essential for catalysis. NMR res ults show that this guanine forms a sheared G . A base pair. The cytos ine residue immediately 5' to the cleavage site forms an AH(+). C base pair with an adenine whose pK(a) is shifted to 6.2 to allow partial p rotonation near neutral pH. Although the residues flanking the cleavag e site are stacked in an A-form pattern, the phosphodiester backbone n ext to the cleavage site on the 3' side is splayed apart. This places the following base-a uracil-in the expanded major groove, The conforma tional flexibility and the lack of steric hindrance of the uracil as w ell as the unoccupied Watson-Crick positions on the sheared G . A base pair can allow loop A to specifically interact with the catalytic dom ain (loop B) without drastically changing its own conformation. The th ree-dimensional structure of loop A provides explanations for previous ly published mutation and structural mapping results.