Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis

Alexander A. Szewczak, Elaine R. Podell, Philip C. Bevilacqua, Thomas R. Cech

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Abstract

The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic folding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure. We have employed temperature 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 °C and is completely unfolded by 60 °C. Calculated thermodynamic parameters for folding of P4-P6 are ΔH°' = -28 ± 3 kcal/mol and ΔS°' = -91 ± 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfate and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary structure is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetrahymena group I intron self-splicing, which have positive or zero ΔH terms, P4-P6 tertiary structure formation is stabilized by a negative ΔH term. This implies that enthalpically favorable hydrogen bond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain.

Original languageEnglish (US)
Pages (from-to)11162-11170
Number of pages9
JournalBiochemistry
Volume37
Issue number32
DOIs
Publication statusPublished - Aug 11 1998

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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