THERMODYNAMIC STABILITY OF THE P4-P6 DOMAIN RNA TERTIARY STRUCTURE MEASURED BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic fol ding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure, We have employed tem perature gradient gel electrophoresis (TGGE) to examine the unfolding of the tertiary structure of P4-P6. In 0.9 mM Mg2+ the global tertiary fold of the molecule has a melting temperature of approximately 40 de grees C and is completely unfolded by 60 degrees C, Calculated thermod ynamic parameters for folding of P4-P6 are Delta H degrees' = -28 +/- 3 kcal/mol and Delta S degrees' = -91 +/- 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfa te and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary stru cture is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetr ahymena group I intron self-splicing, which have positive or zero Delt a H terms, P4-P6 tertiary structure formation is stabilized by a negat ive Delta H term. This implies that enthalpically favorable hydrogen b ond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain .