期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 298, 期 3, 页码 539-555出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1006/jmbi.2000.3691
关键词
footprinting; hairpin ribozyme; lanthanide fluorescence; non-specific metal ion binding; outer-sphere cation binding pocket
Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes inner-sphere coordination to RNA as an obligatory role for metal ions in catalysis. Hence, the hairpin ribozyme is a unique model to study the role of outer-sphere coordinated cations in folding of a catalytically functional RNA structure. Here, we demonstrate that micromolar concentrations of a deprotonated aqueous complex of the lanthanide metal ion terbium(III), Tb(OH)(aq)(2+), reversibly inhibit the ribozyme by competing for a crucial, yet non-selective cation binding site. Tb(OH)(aq)(2+) also reports a likely location of this binding site through backbone hydrolysis, and permits the analysis of metal binding through sensitized luminescence. We propose that the critical cation-binding site is located at a position within the catalytic core that displays an appropriately-sized pocket and a high negative charge density. We show that cationic occupancy of this site is required for tertiary folding and catalysis, yet the site can be productively occupied by a wide variety of cations. It is striking that micromolar Tb(OH)(aq)(2+) concentrations are compatible with tertiary folding, yet interfere with catalysis. The motif implicated here in cation-binding has also been found to organize the structure of multi-helix loops in evolutionary ancient ribosomal RNAs. Our findings, therefore, illuminate general principles of non-selective outer-sphere cation binding in RNA structure and function that may have prevailed in primitive ribozymes of an early RNA world. (C) 2000 Academic Press.
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