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

Research output: Contribution to journalArticle

28 Citations (Scopus)

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
StatePublished - Aug 11 1998

Fingerprint

Denaturing Gradient Gel Electrophoresis
Electrophoresis
Thermodynamics
Thermal gradients
Introns
Thermodynamic stability
Gels
RNA
Tetrahymena
Temperature
Guanosine
Freezing
Melting point
Hydrogen
Hydrogen bonds
Nucleotides
Molecules
Kinetics
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Szewczak, Alexander A. ; Podell, Elaine R. ; Bevilacqua, Philip C. ; Cech, Thomas R. / Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis. In: Biochemistry. 1998 ; Vol. 37, No. 32. pp. 11162-11170.
@article{499c9b29d62849e88a0e5311867e6713,
title = "Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis",
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.",
author = "Szewczak, {Alexander A.} and Podell, {Elaine R.} and Bevilacqua, {Philip C.} and Cech, {Thomas R.}",
year = "1998",
month = "8",
day = "11",
doi = "10.1021/bi980633e",
language = "English (US)",
volume = "37",
pages = "11162--11170",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

}

Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis. / Szewczak, Alexander A.; Podell, Elaine R.; Bevilacqua, Philip C.; Cech, Thomas R.

In: Biochemistry, Vol. 37, No. 32, 11.08.1998, p. 11162-11170.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis

AU - Szewczak, Alexander A.

AU - Podell, Elaine R.

AU - Bevilacqua, Philip C.

AU - Cech, Thomas R.

PY - 1998/8/11

Y1 - 1998/8/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0032508366&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032508366&partnerID=8YFLogxK

U2 - 10.1021/bi980633e

DO - 10.1021/bi980633e

M3 - Article

C2 - 9698362

AN - SCOPUS:0032508366

VL - 37

SP - 11162

EP - 11170

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 32

ER -