Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

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.

Single-molecule studies of riboswitch folding

“…This finding raises the possibility that one role of adenine may be to restrict the states available for folding. Supporting this interpretation, a constitutively “on” mutant of the add riboswitch also exhibited a lower variation in average dwell times in the absence of ligand, although the reduction was only 2-fold in that case [56]. However, for a different purine riboswitch (xpt guanine, section 4.3 ), the variation in dwell times for the docked and undocked states was also a factor of ~100 [55], but the addition of ligand had no substantial effect on that variation.…”

“…Typically, a riboswitch aptamer domain is site-specifically labeled with both donor and acceptor fluorophores for FRET, then attached to a glass (or quartz) surface via linker chemistry (Figure 2) [51]. To date, lysine [52], SAM class I [53], SAM class II [54], guanine [55], adenine [56–58], TPP [59], c-di-GMP class I [60], and preQ 1 class I & II riboswitches [61, 62] have been explored using smFRET methodologies (section 4 ).…”

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

“…This analysis assumes that the U-to-A binding-site mutation displaces the glycine without stabilizing the binding site in a "bound-like" conformation. As a counter-example, the C64U mutant of the adenine riboswitch fails to respond to ligand, instead causing constitutive activation of the downstream gene (Tremblay et al 2011). Given the significant disruption of dimerization when both binding sites are mutated, and the similarity of U78A/U207A's dimer affinity to that of the WT constructs in the absence of ligand (Fig.…”

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…N-Methylisatoic anhydride dissolved in dimethyl sulfoxide was added and incubated for 80 min at 37°C. N-Methylisatoic anhydride-treated RNA was precipitated, resuspended, and reverse transcribed using the oligonucleotide BtuB202 (Table 3) as described previously (31). ␤-Galactosidase Assays-␤-Galactosidase experiments were performed as described previously (32).…”

“…Selective 2Ј-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE) Analysis-SHAPE was performed as described previously (31). RNA samples were slowly cooled in a solution containing 100 mM K-HEPES, pH 8.0, and 100 mM NaCl.…”