Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation.
For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the nature of the native state ensemble, the mechanisms of unfolding, and the stability of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combining chemical denaturation and hydroxyl radical footprinting methods. We have determined unfolding isotherms for each of 26 discrete sites of protection located throughout the Tetrahymena thermophila group I ribozyme. The cooperativity of folding, m-value, and the free energy, DeltaG degrees N-U, associated with formation of each tertiary contact was determined by analysis of the isotherms. The DeltaG degrees N-U values measured in this study vary from 1.7 +/- 0.2 to 7. 6 +/- 1.2 kcal mol-1. Thus, the stability of these discrete tertiary contacts vary by almost 104. In addition, an intradomain contact and three interdomain contacts show high cooperativity (m-values of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol-1 M-1) indicating that these contacts exhibit global cooperatively in their folding behavior. This new approach to examining RNA stability provides an exciting comparison to our understanding of protein structure and folding mechanisms.
Ralston
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Center for Synchrotron Biosciences, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
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Brenowitz
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Chance
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D000495:Allosteric Site; D000818:Animals; D001483:Base Sequence; D017665:Hydroxyl Radical / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D020871:RNA Stability / Q000187:drug effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012997:Solvents; D013696:Temperature; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000235:genetics*; D013816:Thermodynamics; D017186:Titrimetry; D014508:Urea / Q000494:pharmacology
D016337:RNA, Catalytic; D012997:Solvents; D017665:Hydroxyl Radical; D014508:Urea; D008274:Magnesium