Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

被引:226
作者
Kim, HD
Nienhaus, GU
Ha, T
Orr, JW
Williamson, JR
Chu, S [1 ]
机构
[1] Stanford Univ, Dept Appl Phys & Phys, Stanford, CA 94305 USA
[2] Univ Ulm, Dept Biophys, D-89069 Ulm, Germany
[3] Univ Illinois, Dept Phys, Urbana, IL 61801 USA
[4] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA
[5] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
关键词
D O I
10.1073/pnas.032077799
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 305 ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.
引用
收藏
页码:4284 / 4289
页数:6
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