Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA

Abstract

In Gram-positive bacteria, T-box riboswitches regulate the expression of aminoacyl-tRNA synthetases and other proteins in response to fluctuating transfer RNA aminoacylation levels under various nutritional states. T-boxes reside in the 5'-untranslated regions of the messenger RNAs they regulate, and consist of two conserved domains. Stem I contains the specifier trinucleotide that base pairs with the anticodon of cognate tRNA. 3' to stem I is the antiterminator domain, which base pairs with the tRNA acceptor end and evaluates its aminoacylation state. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition by this riboswitch remains ill defined. Here we demonstrate that the ~100-nucleotide T-box stem I is necessary and sufficient for specific, high-affinity (dissociation constant (Kd) ~150 nM) tRNA binding, and report the structure of Oceanobacillus iheyensis glyQ stem I in complex with its cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins such as the ribosome, or proteins such as aminoacyl-tRNA synthetases, but is unprecedented for a compact mRNA domain. The C-shaped stem I cradles the L-shaped tRNA, forming an extended (1,604 Å(2)) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif of RNase P and the L1 stalk of the ribosomal E-site. Because these ribonucleoproteins and T-boxes are unrelated, this strategy to recognize a universal tRNA feature probably evolved convergently. Mutually induced fit of stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation.

Figure 1. Overall structure of the T-Box Stem I in complex with tRNA

a, ITC analysis of tRNA binding by full-length glyQS T-box (residues 14-182). b, Binding by a 3’-truncated T-box (residues 14-158). c, Binding by an isolated T-box Stem I (residues 14-113). d, Sequence and secondary structure of the cocrystallized glyQ Stem I and tRNA^Gly RNAs. Leontis-Westhof symbols denote non-canonical base pairs. Lines with embedded arrowheads denote chain connectivity. The tRNA (shaded) is numbered conventionally (‘t’ precedes tRNA residues). e. Cartoon of the complex structure. Color-coding as in (a); segments altered to facilitate crystallization and the YbxF protein are in white.

Figure 2. Interactions between Stem I and tRNA

a, Specifier-anticodon interaction. Dashed lines denote hydrogen bonds. Displacement of A86 forms a pocket (dashed oval). b, The two interdigitated T-loops at the distal end of Stem I stack on the tRNA elbow. c, Stacking of the apical Stem I base triple on the tRNA elbow. Yellow dashed lines denote tRNA elbow base-pairing. d, tRNA residue tU20 flips out to stack with the Stem I C64 ribose (van der Waals surfaces of interacting residues shown). e. Mutagenesis and ITC analysis of selected Stem I-tRNA interactions. Error bars denote s.e.m. (n≥2).

Figure 3. Induced fit of tRNA by Stem I binding

a, Superposition of ASL of free tRNA^Gly (PDB 2LBJ, orange) with the cocrystal structure. Steric clash between the free structure and the loop E motif (green) is evident. b, Superposition of the ASL of free tRNA^Phe (PDB 1EHZ, orange) with the cocrystal structure. Note extrusion of tU33. c, Comparison of Stem I-bound tRNA^Gly with free tRNA^Phe (orange) seen from the direction of the elbow. The approximate location of the hinge at the t26βt44 pair is indicated. d, Comparison of Stem I-bound tRNA^Gly with the ribosome-bound P/P tRNA (PDB 4GD2, orange).

Figure 4. Stem I reorganization by tRNA binding

a, Superposition of an isolated Stem I distal fragment (PDB 4JRC, black) and the complex. Clash between the helix (partly shown as spheres) of the fragment and the tRNA is relieved by flexing below the T-loops in the complex. b, Superposition of the specifier free (PDB 2KZL, orange) and bound to tRNA. Specifier nucleotides rotate outward, and A90 is displaced. c, Superposition of an isolated Stem I proximal fragment (PDB 2KZL, orange) and the complex. tRNA binding induces bending of Stem I (arrow) bringing its 3’-terminus ~50 Å closer to the acceptor end of tRNA.