Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

JOURNAL OF MOLECULAR BIOLOGY

卷:386

Article

Chauhan, Seema²  Behrouzi, Reza¹  Rangan, Prashanth¹  Woodson, Sarah A.¹ 

[1] Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA

[2] Johns Hopkins Univ, Dept Chem, Baltimore, MD 21218 USA

关键词: RNA folding;    ribozynie;    stopped-flow fluorescence;    time-resolved footprinting;    2-aminopurine;   

DOI  :  10.1016/j.jmb.2008.12.075

来源: Elsevier

【 摘 要 】

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (tau<30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg2+, Consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1. s (1) at 37 degrees C. From comparison with time-resolved Fe(IT)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I-C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme. (C) 2009 Elsevier I.td. All rights reserved.

【 授权许可】

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