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Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

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Author(s)

Renaud Tremblay, Jean-Francois Lemay, Simon Blouin, Jerome Mulhbacher, Eric Bonneau, Pascale Legault, Paul Dupont, Carlos Penedo, Daniel A. Lafontaine

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Abstract

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

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Original languageEnglish
Pages (from-to)27406-27415
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number31
DOIs
Publication statusPublished - 5 Aug 2011

    Research areas

  • RESONANCE ENERGY-TRANSFER, 4-WAY DNA JUNCTION, SAM-I RIBOSWITCH, APTAMER DOMAIN, PURINE RIBOSWITCH, BACILLUS-SUBTILIS, RNA STRUCTURE, ADENINE RIBOSWITCH, STRUCTURAL BASIS, GENE-EXPRESSION

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