270 MHz 1H-MNR Studies of four “branched” tetraribonucleotides: A3'p5'A 2'p5'G3'p5'U, A3'p5'A 2'p5'G'3'p5'U, U3'p5'A 2'p5'G3'p5'U & U3'p5'A 2'p5'G3'p5'C which are formed as the lariat branch-point in the pre-mRNA processing reactions (splicing)

Abstract

1H-NMR conformational studies of four title branched ribonucleotides, formed as a local structure in the splicing intron, have been carried out by assigning 1H resonances by 2D NMR such as triple Relay-COSY and 31P/ 1H correlation experiments and then observing the temperature-dependent (7 – 80 °C) (i) chemical shifts of aromatic and sugar protons, and (ii) shifts of N /af S equilibrium of different sugar moieties. Detailed 2D NOE (NOESY) experiments have subsequently furnished informations regarding the spatial proximities of different protons in these molecules. These studies suggest a coherent conformational picture of the four title branched tetraribonucleotides: (a) the branch-point adenosine (A*) and its 3'-terminal pyrimidine residue are N-N stacked; (b) the A* is coplanar with its 2'-terminal guanosine residue; (c) the nucleotide (A or U) at the 5'-terminus of the A* is S-S stacked with the 2'-terminal guanosine residue and it is also S-N stacked to the A*; (d) the glycosidic bonds of the 3'-terminal pyrimidine residue (C or U) and 5'-terminal residue (A) are in anti and syn conformations respectively, whereas the A* and the 2'-terminal guanosine residues have approximately an equal population of syn and anti conformers. These conformational features of branched tetraribonucleotides are reminiscent of a distorted linear 3' → 5' linked single stranded A-RNA helix fragment.