THE DETAILED STRUCTURE OF TANDEM G-A MISMATCHED BASE-PAIR MOTIFS IN RNA DUPLEXES IS CONTEXT-DEPENDENT

The solution structure of the RNA duplex (rGGGCUGAAGCCCU), containing tandem G.A mismatches has been determined by NMR spectroscopy and rest rained molecular dynamics. A homonuclear 3D TOCSY-NOESY was used to de rive 18 to 30 distance restraints per nucleotide, as well as all gamma torsion angles and sugar puckers for the central UGAA part of the mol ecule. Using these constraints, together with cross-strand distances, involving exchangeable imino protons, and essentially all other torsio n angles that can accurately be determined (i.e. beta, epsilon) otherw ise, the structure of the UGAA domain could be determined with high pr ecision (r.m.s.d. 0.62 Angstrom), without the aid of isotopically enri ched RNA. The G.A base-pairs are of the sheared pairing type, with bot h nucleotides in the anti conformation, and hydrogen bonds between the guanine 2-amino and the adenine N7 and between the guanine N3 and the adenine 6-amino. Surprisingly the sugar of the guanosine of the G.A m ismatch adopts a 2'-endo sugar pucker conformation. Comparison with ot her RNA structures, in which two such G.A base-pairs are formed reveal s that this detailed structure depends on the identity of the base 5' to the guanosine in the tandem G.A base-pairs. A geometrical model for the incorporation of sheared tandem G.A base-pairs in A-form helices is formulated, which explains the distinct different stacking properti es and helical parameters in sequences containing tandem sheared G.A b ase-pairs. (C) 1997 Academic Press Limited.