Aa. Szewczak et al., THERMODYNAMIC STABILITY OF THE P4-P6 DOMAIN RNA TERTIARY STRUCTURE MEASURED BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS, Biochemistry, 37(32), 1998, pp. 11162-11170
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
.