| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 88 === We have determined the solution structure of a TCC-loop hairpin in the cruciform promoter for the bacteriophage N4 virion RNA polymerase (N4 vRNAP). This hairpin and its complementary GGA-loop hairpin are extruded at physiological superhelical density and are required for vRNAP recognition. Contrary to its complementary GGA-loop, the three pyrimidines in the TCC-loop are all 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 stabilize the hairpin structure. The resulting structures display extensive loop base-stacking rearrangement yet minor backbone distortion, which is largely accomplished through some loop ζ and α torsional angle changes. Consistent with the structural studies, UV melting of the GAAGCTCCGCTTC hairpin revealed a higher melting temperature (66 °C) than that of the GAACGTCCCGTTC hairpin (58 °C) with reversed stem G·C base-pairs, indicating significant contribution from the extra three loop-stem H-bonds. Thermodynamic parameters ΔG°25 of the GAAGC TCC GCTTC hairpin and its complementary GAAGC GGA GCTTC hairpin are -4.1 and -4.3 kcal/mol respectively, indicating approximately equal contribution of each hairpin to the cruciform formation of the N4 virion RNA polymerase promoter. No significant loop dynamics in the microsecond to millisecond NMR time-scale was observed, and the abundant well-defined exchangeable and non-exchangeable proton NOEs allowed us to efficiently determine a well-converged family for the final structures of the TCC-loop hairpin.
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