Formation of Tertiary Interactions during rRNA GTPase Center Folding

JOURNAL OF MOLECULAR BIOLOGY

卷:427

Article

Rau, Michael J.¹  Welty, Robb¹  Stump, W. Tom¹  Hall, Kathleen B.¹ 

[1] Washington Univ, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 USA

关键词: 2-aminopurine;    stopped-flow fluorescence;    RNA folding kinetics;    Mg2+-dependent RNA folding;    NMR;   

DOI  :  10.1016/j.jmb.2015.07.013

来源: Elsevier

【 摘 要 】

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 N-15-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. (C) 2015 Elsevier Ltd. All rights reserved.

【 授权许可】

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