COMMON STRUCTURAL FEATURES OF UUCG AND UACG TETRALOOPS IN VERY SHORT HAIRPINS DETERMINED BY UV ABSORPTION, RAMAN, IR AND NMR SPECTROSCOPIES

Thermodynamic and structural properties of two UNCG tetraloops in very short hairpin octamers, 5'-r(GCUUCGGC)-3' and 5'-r(GCUACGGC)-3', have been studied by means of various physical techniques. Melting profile s of both octamers, obtained from UV absorption spectra taken as a fun ction of temperature, are consistent with a monophasic, progressive an d completely reversible order-to-disorder transition and confirm their unusual structural stability (T-m>51 degrees C). The H-1, C-13 and (3 1)p NMR chemical shifts and coupling constants of the UACG loop nucleo tides are comparable with those reported previously for UUCG loops, i. e. 2'-endo/anti conformation of the second and third nucleotide of the loop as well as the syn orientation of the ultimate guanine base and the A-type double helical conformation of the hairpin stem. Simulation of quantitative NOESY volumes shows that the UACG octamer adopts a ve ry rigid compact structure which is well represented by an average ord er parameter of 0.9. Three base-pairs and four additional strong hydro gen bonds are undoubtedly responsible for such limited flexibility. Ra man and infrared spectra as a function of temperature reflect the orde r-to-disorder transition, as well. Vibrational conformational markers in low temperature spectra of both octamers indicate the hairpin struc ture as the major conformer in aqueous phase. These spectra further su pport the structural features of most of the nucleotides involved in t he tetraloops and clearly demonstrate the structural similarities of t he phosphodiester backbone in both hairpins. Consequently, on the basi s of all present results, one can deduce that the conformational featu res of the UUCG and UACG tetraloops seem to be inherent to the UNCG ty pe tetraloops, regardless of either the nature of the tetraloop second base or the stem length.