NMR ASSIGNMENT OF A 2'-HYDROXYL PROTON FROM THE UUCG TETRALOOP THROUGH LONG-RANGE CORRELATIONS WITH C-13

The conformation and hydrogen bonding properties of the slowly exchang ing U5 2'-hydroxyl group in the UUCG RNA tetraloop hairpin 5'[GGAC(UUC G)GUCC]3' were investigated by heteronuclear NMR spectroscopy. Proton- carbon coupling constants for 2'-OH to C1', C2' and C3' were measured for the four nucleosides (adenosine, cytidine, guanosine and uridine) in dimethyl sulfoxide, The coupling constants ranged from 1.5 to 4.0 H z. For the UUCG hairpin similar couplings were used to assign definiti vely the U5 2'-OH resonance, The relative magnitudes of the coupling c onstants to C1' and C3' indicate that the U5 2'-OH adopts a trans conf ormation with respect to U5 C3'. The observation of a strong 2'-OH to H1' NOE and no 2'-OH to H3' NOE confirms the conformational preference of this 2'-OH, The low exchange rate with solvent (1.2 +/- 0.1 s(-1) at 10 degrees C in buffer containing 100 mu M EDTA, 50 mM NaCl and 10 mM sodium phosphate, pH 6.1) and the low chemical shift vs, temperatur e coefficient of the 2'-OH proton resonance (-0.004 +/- 0.001 ppm degr ees C-1) indicate that the hydroxyl group is strongly protected from e xchange and probably acts as a hydrogen bond donor, The results are in terpreted in the context of previous structural studies of the UUCG te traloop and provide a basis for comparison to slowly exchanging hydrox yl protons in other RNA molecules.