Abstract
The non-exchangeable 1H-NMR signals of the branch core trinucleotide of the lariat branch site (A2′p5′G 3′p5′C), 1) and its derivatives 2 and 3 are completely assigned using one- and two- dimensional NMR techniques including NOE, COSY, NOESY, 1H-1HINADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T1 measurements, the following properties of the trimers are deduced.(i)The unique stacking behavior of the trimers is S1′N 3′N, and the sugar rings exist predominantly in the N-conformation (3′-endo-2′-exo).(ii)The sugar-base orientations appear to be anti.(iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21°C and pH 7.2.(iv) The calculated values for the torsion angles εt andγ+ for the trimers are 201–203° and 71–86%, respectively, while the percent β1 values are higher for the guanine (87–92%) than the cytosine residues (73–77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.