Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme

Takeshi Uchida, Keiji Takamoto, Qin He, Mark R. Chance, Michael D. Brenowitz

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Synchrotron hydroxyl radical (·OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg2+-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200mM NaCl and folded by the addition of 10mM MgCl2, multiple kinetic phases are observed for ·OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, ·OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.

Original languageEnglish (US)
Pages (from-to)463-478
Number of pages16
JournalJournal of Molecular Biology
Volume328
Issue number2
DOIs
StatePublished - Apr 25 2003

Fingerprint

Tetrahymena thermophila
Catalytic RNA
Ions
Catalytic Domain
Buffers
Monovalent Cations
Synchrotrons
Magnesium Chloride
Divalent Cations
Hydroxyl Radical
Salts
RNA

Keywords

  • Footprinting
  • Hydroxy radical
  • RNA folding
  • Synchrotron

ASJC Scopus subject areas

  • Virology

Cite this

Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme. / Uchida, Takeshi; Takamoto, Keiji; He, Qin; Chance, Mark R.; Brenowitz, Michael D.

In: Journal of Molecular Biology, Vol. 328, No. 2, 25.04.2003, p. 463-478.

Research output: Contribution to journalArticle

Uchida, Takeshi ; Takamoto, Keiji ; He, Qin ; Chance, Mark R. ; Brenowitz, Michael D. / Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme. In: Journal of Molecular Biology. 2003 ; Vol. 328, No. 2. pp. 463-478.
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abstract = "Synchrotron hydroxyl radical (·OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg2+-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200mM NaCl and folded by the addition of 10mM MgCl2, multiple kinetic phases are observed for ·OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90{\%} burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100{\%} burst phase of isolated P4-P6 at 200mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, ·OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.",
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