An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

Abstract

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In >50% of the glycine riboswitches, the leader-linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader-linker interaction improved the glycine-binding affinities by 4.5-to 86-fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand-binding cooperativity in the glycine riboswitch when the leader-linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.

Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis

“…In the presence of glycine, which binds to the riboswitch, the terminator appears to be destabilized and consequently~70 % of the transcript is full-length (Mandal et al, 2004). Glycine-dependent activation of this riboswitch has also been reported in other species (Sherman et al, 2012). We hypothesize that s B -dependent formation of glycine is responsible for activating transcription of the gcvT operon via this riboswitch, as we found lmo2539 (glyA; Group B, Table S2), which encodes a serine hydroxymethyltransferase that catalyses the interconversion of L-serine to glycine (Stauffer, 1996), to be positively regulated by s B .…”

“…As one mechanism for post-transcriptional regulation is through riboswitches, we further evaluated the 59 UTR Mandal et al (2004); the leader-linker interaction is based on the data for lmo1348 provided by Sherman et al (2012). The terminator was identified using TransTermHP (Kingsford et al, 2007).…”

“…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).…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…When in water only, RNA is expected to be unstructured, and W-band spectrum ( Figure 4C top and S8) with a SNR value of 18:1 reveal disorder, with spectral simulation giving a correlation time of 0.4 ns. It has previously been shown that, upon addition of salts and ligands, the VC glycine riboswitch folds with formation of the kink turn [53,54,63,64] where, based upon comparisons to X-ray models of other k-turn motifs [65,66], the modified U2-base is expected to become base paired/base stacked. Hence, a decrease in dynamics (an increase in correlation time) is anticipated.…”

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

“…Recently, two groups have independently provided a possible explanation for the unusually high divalent cation requirement of this RNA. Kladwang et al (2012) and Sherman et al (2012) found that the linker segment connecting the two aptamer domains, rather than being single-stranded, forms a hitherto unrecognized helix (P0 hereafter) with a conserved RNA segment 5 ′ to the first aptamer domain (Fig. 1A).…”