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Effects of polyvalent cations on the folding of an rRNA three-way junction and binding of ribosomal protein S15

Published online by Cambridge University Press:  01 August 1998

ROBERT T. BATEY  and
JAMES R. WILLIAMSON
ROBERT T. BATEY
Affiliation:
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
JAMES R. WILLIAMSON
Affiliation:
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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Abstract

The Bacillus stearothermophilus ribosomal protein S15 binds to a phylogenetically conserved three-way junction formed by the intersection of helices 20, 21, and 22 of eubacterial 16S ribosomal RNA, inducing a large conformational change in the RNA. Like many RNA structures, this three-way junction can also be folded by the addition of polyvalent cations such as magnesium, as demonstrated by comparing the mobilities of the wild-type and mutant junctions in the absence and presence of polyvalent cations in nondenaturing polyacrylamide gels. Using a modification interference assay, critical nucleotides for folding have been identified as the phylogenetically conserved nucleotides in the three-way junction. NMR spectroscopy of the junction reveals that the conformations induced by the addition of magnesium or S15 are extremely similar. Thus, the folding of the junction is determined entirely by RNA elements within the phylogenetically conserved junction core, and the role of Mg2+ and S15 is to stabilize this intrinsically unstable structure. The organization of the junction by Mg2+ significantly enhances the bimolecular association rate (kon) of S15 binding, suggesting that S15 binds specifically to the folded form of the three-way junction via a tertiary structure capture mechanism.

Keywords

Bacillus stearothermophilusmagnesium-dependent RNA foldingmodification interference analysisNMRribosome16S RNA
Type
Research Article
Information
RNA , Volume 4 , Issue 8 , August 1998 , pp. 984 - 997
Copyright
© 1998 RNA Society

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