Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

Renaud Tremblay, Jean-François Lemay, Simon Blouin, Jérôme Mulhbacher, Éric Bonneau, Pascale Legault, Paul Dupont, J. Carlos Penedo, Daniel A. Lafontaine
2011 Journal of Biological Chemistry  
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
more » ... e-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.
doi:10.1074/jbc.m111.229047 pmid:21676871 pmcid:PMC3149334 fatcat:urripi6zxvh7jjd5zs3rzlie2a