title: Formation of Tertiary Interactions during rRNA GTPase Center Folding.
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 Mg(2+) ions, which is stabilized by the L11 protein in cocrystal structures. Here, we monitor the kinetics of its tertiary folding and Mg(2+)-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 Mg(2+) 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 4s and with global folding from 1 to 5s. Site-specific substitution with (15)N-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 Mg(2+) and suggest that there are two initial sites of Mg(2+) ion association.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, MO 63110, USA. Electronic address: mjrau01@gmail.com.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rau
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, MO 63110, USA. Electronic address: robb@wustl.edu.
authors.forename: Robb
authors.initials: R
authors.lastname: Welty
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, MO 63110, USA. Electronic address: stump@biochem.wustl.edu.
authors.forename: W
authors.initials: W
authors.lastname: Tom Stump
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University Medical School, St. Louis, MO 63110, USA. Electronic address: kathleenhal@gmail.com.
authors.forename: Kathleen B
authors.initials: KB
authors.lastname: Hall
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism* / Q000648:ultrastructure; D001411:Geobacillus stearothermophilus / Q000235:genetics; D006860:Hydrogen Bonding; D008274:Magnesium / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D020124:Thermotoga maritima / Q000235:genetics
chemical_list: D012338:RNA, Ribosomal, 23S; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D020558:GTP Phosphohydrolases; D008274:Magnesium
keywords: 2-aminopurine; Mg(2+)-dependent RNA folding; NMR; RNA folding kinetics; stopped-flow fluorescence