Formation of Tertiary Interactions during rRNA GTPase Center Folding

Michael J. Rau, Robb Welty, W. Tom Stump, Kathleen B. Hall

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

The 60-nt GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2 + ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg2 +-dependent intermediate states by observing selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine replaced three individual adenines, two of which make long-range stacking interactions and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2 + addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site specific, with local conformational changes occurring from 5 ms to 4 s and with global folding from 1 to 5 s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2 + and suggest that there are two initial sites of Mg2 + ion association.

Original languageEnglish
Pages (from-to)2799-2815
Number of pages17
JournalJournal of Molecular Biology
Volume427
Issue number17
DOIs
StatePublished - Aug 17 2015

Keywords

  • 2-aminopurine
  • Mg-dependent RNA folding
  • NMR
  • RNA folding kinetics
  • stopped-flow fluorescence

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