Stable formation of a pyrimidine-rich loop hairpin in a cruciform promoter

We have determined the solution structure of a TCC-loop hairpin in the cruc iform promoter for the bacteriophage N4 virion RNA polymerase (N4 vRNAP). T his hairpin and its complementary GGA-loop hairpin are extruded at physiolo gical superhelical density and are required for vRNAP recognition. Contrary to its complementary GGA-loop, the three pyrimidines in the TCC-loop are a ll unpaired. However, with the help of two juxtaposed stem Watson-Crick G.C base-pairs, each nucleotide in the loop employs a special method to stabil ize the hairpin structure. The resulting structures display extensive loop base-stacking rearrangement yet minor backbone distortion, which is largely accomplished through some loop zeta and or torsional angle changes. Consis tent with the structural studies, UV melting of the GAAGCTCCGCTTTC hairpin revealed a higher melting temperature (66 degrees C) than that of the GAACG TCCCGTTC hairpin (58 degrees C) with reversed stem G.C base-pairs, indicati ng significant contribution from Be extra three loop-stem H-bonds. Thermody namic parameters Delta G degrees(25), of the GAAGCTCCGCTTC hairpin and its complementary GAAGCGGAGCTTC hairpin are -4.1 and -4.3 kcal/mol respectively , indicating approximately equal contribution of each hairpin to the crucif orm formation of the N4 virion RNA polymerase promoter. No significant loop dynamics in the microsecond to millisecond NMR time-scale was observed, an d the abundant well-defined exchangeable and nonexchangeable proton NOEs al lowed us to efficiently determine a well-converged family for the final str uctures of the TCC-loop hairpin. (C) 1999 Academic Press.