Anomalous Conformations of RNA Constituents : 2D NMR and Calculational Studies

Abstract

Short single-stranded RNA fragments normally display a strong tendency to favour a right-handed helical conformation. Relatively small changes in enthalpy and/or entropy of stacking, induced by minor structural variations may cause a large difference in stacking behaviour in aqueous solution, but do not appear to affect the detailed geometry of the stacked state. Recently developed NMR methods that allow for a determination of the sugar-phosphate backbone geometry along β (O5’-C5’), γ (C5’-C4’), δ (C4’-C3’), and ε (C3’-O3’) are surveyed. At the level of trimers and higher oligomers conformational transmission factors, such as next-nearest-neighbour interactions, may come into play. For example, the trimer U-A¯-U (A¯ = m₂ ⁶A) behaves in a fashion that can be predicted from the known stacking properties of its dimer constituents U-A¯ and A¯-U, whereas the trimer A¯-U-A¯ behaves in an entirely different way. In the latter compound the two purines engage in a 1-3 stacking interaction. At the same time the central pyrimidine residue is pushed outside the purine-purine interaction zone (bulge-out).

Several interesting properties of bulges have come to light: (i) a longer alternating pu-py sequence displays multiple bulges, witness A¯-U-A¯-U-A¯; (ii) a strong stacking interaction at its 3’-end does not affect the bulge, for example in A¯-U-A¯-U; (iii) in contrast, the bulge is abandoned in favour of a normal right-handed stacking pattern by a strong stacking interaction at its 5’-end: A¯-A¯-U-A¯; (iv) a self-complementary alternating pu-py sequence, e.g. (A-U)₃, is able to convert from a bulged single strand at elevated temperatures into a regular A-type duplex at low temperature. Thus, bulge-out structures may occur either in loops or under conditions where a duplex is forced to open up.

Another interesting conformation is shown by the 3A’-terminal C¯-A¯ in C¯-C¯-A¯ (C¯ = m₂ ⁴C), a chemically modified 3’-acceptor of tRNAs. In contrast to the usual right-handed parallel stacking pattern favoured by the C¯-C¯ part, the 3’-terminal A¯ residue prefers to adopt a left-handed antiparallel stacking.

With the aid of molecular-mechanics calculations (AMBER program) various plausible A-U-A and C-C-A models could be generated.