Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

Abstract

The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.

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

“…This particular glycine riboswitch controls the VC1422 gene, which encodes a sodium/alanine-or glycine-symporter. It was the initial glycine riboswitch characterized ) and has served as the prototype for subsequent biochemical study (Lipfert et al 2007;Kwon and Strobel 2008;Erion and Strobel 2011). For clarity, the positional nucleotide numbering remains consistent with previous reports, with the leader nucleotides assigned positions -7 to -1 (Sherman et al 2012;Esquiaqui et al 2014).…”

Modulation of quaternary structure and enhancement of ligand binding by the K-turn of tandem glycine riboswitches

“…The previously reported ability of tandem glycine aptamers lacking the P0 K-turn to fold and bind glycine in vitro (Mandal et al 2004;Lipfert et al 2007;Kwon and Strobel 2008;Huang et al 2010;Lipfert et al 2010;Butler et al 2011;Erion and Strobel 2011) is reminiscent of the results of truncation of the hammerhead and hairpin ribozymes. In studies of the former, efforts to generate a kinetically well-behaved minimal ribozyme yielded an RNA construct amenable to biochemical and structural characterization (for review, see Blount and Uhlenbeck 2005).…”

“…Crystallographic structure determination demonstrates that isolated glycine riboswitch aptamer domains adopt the same fold as in tandem (Huang et al 2010;Butler et al 2011). Moreover, both in solution and in crystals, the isolated aptamer domains show a propensity to oligomerize, utilizing what appears to be the same interface as that used by the tandem aptamers when present in a single RNA chain (Huang et al 2010;Butler et al 2011;Erion and Strobel 2011).…”

“…The unusually compact F. nucleatum tandem glycine riboswitch has been the subject of multiple biochemical and structural studies (Kwon and Strobel 2008;Butler et al 2011;Erion and Strobel 2011;Kladwang et al 2012;Sherman et al 2012). Indeed, the only available crystal structure of a tandem glycine riboswitch is for the RNA from this organism (Butler et al 2011).…”

Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis

“…3). This glycine riboswitch is conserved across many bacterial species and occurs upstream of genes encoding proteins of the glycine cleavage system, which facilitates use of glycine as an energy source (Butler et al, 2011;Erion & Strobel, 2011;Huang et al, 2010;Kwon & Strobel., 2008;Mandal et al, 2004;Sherman et al, 2012). In Bacillus subtilis, it has been shown that this riboswitch regulates transcription of the downstream gcvT operon (Mandal et al, 2004;Phan & Schumann, 2007).…”