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. 2012 Apr;18(4):759-70.
doi: 10.1261/rna.031518.111. Epub 2012 Feb 21.

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

Nathan J Baird  1 , Jinwei Zhang, Tomoko Hamma, Adrian R Ferré-D'Amaré
Affiliations

YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops

Nathan J Baird et al. RNA. 2012 Apr.

Abstract

The archaeal protein L7Ae and eukaryotic homologs such as L30e and 15.5kD comprise the best characterized family of K-turn-binding proteins. K-turns are an RNA motif comprised of a bulge flanked by canonical and noncanonical helices. They are widespread in cellular RNAs, including bacterial gene-regulatory RNAs such as the c-di-GMP-II, lysine, and SAM-I riboswitches, and the T-box. The existence in bacteria of K-turn-binding proteins of the L7Ae family has not been proven, although two hypothetical proteins, YbxF and YlxQ, have been proposed to be L7Ae homologs based on sequence conservation. Using purified, recombinant proteins, we show that Bacillus subtilis YbxF and YlxQ bind K-turns (K(d) ~270 nM and ~2300 nM, respectively). Crystallographic structure determination demonstrates that both YbxF and YlxQ adopt the same overall fold as L7Ae. Unlike the latter, neither bacterial protein recognizes K-loops, a structural motif that lacks the canonical helix of the K-turn. This property is shared between the bacterial and eukaryal family members. Comparison of our structure of YbxF in complex with the K-turn of the SAM-I riboswitch and previously determined structures of archaeal and eukaryal homologs bound to RNA indicates that L7Ae approaches the K-turn at a unique angle, which results in a considerably larger RNA-protein interface dominated by interactions with the noncanonical helix of the K-turn. Thus, the inability of the bacterial and eukaryal L7Ae homologs to bind K-loops probably results from their reliance on interactions with the canonical helix. The biological functions of YbxF and YlxQ remain to be determined.

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Figures

FIGURE 1.
FIGURE 1.
Sequence and biochemical characterization of YbxF and YlxQ. (A) Multiple sequence alignment of B. subtilis YbxF, YlxQ, and their archaeal and eukaryal homologs. (Bs) Bacillus subtilis; (Mj) Methanococcus jannaschii; (Pa) Pyrococcus abyssi; (Sc) Saccharomyces cerevisiae; (Hs) Homo sapiens. Secondary structure elements shared by all six proteins are indicated. Colored bars denote different levels of sequence conservation among the six proteins: (gray) similar; (yellow) highly similar; (dark orange) invariant. (Red dots) YbxF residues that make RNA contacts. (B) Phylogenetic dendrogram calculated using ClustalW2 (Larkin et al. 2007) for the six proteins whose sequences are shown in A. (C) Electrophoretic mobility shift analysis of K-turn and K-loop binding by M. jannaschii L7Ae and B. subtilis YbxF and YlxQ. (D,E) Isothermal titration calorimetric analysis of K-turn binding by B. subtilis YbxF (left) and YlxQ (right).
FIGURE 2.
FIGURE 2.
RNA recognition by YbxF. (A) Secondary structure of the Thermoanaerobacter tengcongensis SAM-I riboswitch construct used for cocrystallization. The distal end of P3 was engineered with the P3ext sequence described in Baird and Ferré-D'Amaré (2010). Base pairs observed in the cocrystal structure of the riboswitch bound to YbxF are denoted using Leontis and Westhof (2001) symbols. Arabic numbers indicate the numbering scheme of the riboswitch aptamer domain. K-turn residues are named using the nomenclature of Liu and Lilley (2006). (Green) K-turn; (red) P4 helix; (blue) pseudoknot. (B) Cartoon representation of YbxF bound to the SAM-I riboswitch K-turn. Secondary structure elements of the protein and several residues of the K-turn are named as in Figure 1A and part A, respectively. A portion of the 2.8 Å resolution anneal-omit 2|Fo| − |Fc| electron density map corresponding to the K-turn is shown contoured at 2σ. (C) Superposition of the SAM-I riboswitch core (red, from RNA complexed to YbxF), the protein-free T. tencongensis riboswitch (Montange et al. 2010) (PDB ID 3GX5, gray), and the B. subtilis yitJ riboswitch (Lu et al. 2010) (PDB ID 3NPB, cyan) demonstrates that P4/L4 is mobile in relation to the body of the aptamer domain. (D) Magnified view of the NC helix from the RNA complexed to YbxF reveals no interactions between G at position 2n and A at position 2b. (E) The protein-free SAM-I riboswitch (3GX5) has the same sequence as in D and indicates formation of the G•A sheared pair between 2b–2n residues of the NC helix. (F) Structural alignment of YbxF (red) and L7Ae (gray). (G) Structural alignment of YbxF (red) and L30e (cyan).
FIGURE 3.
FIGURE 3.
Global comparison of K-turn-binding proteins bound to K-turns. (A) Alignment of K-turn G•A sheared pairs from RNP complexes (YbxF, red; L7Ae, gray) reveal ∼12° counterclockwise rotation of YbxF relative to L7Ae. (B) Eukaryal protein 15.5kD (brown) aligns closely with YbxF in its orientation on the K-turn, while (C) eukaryal protein L30e (cyan) has an intermediate degree of rotation. (D) Analysis of the three interaction surfaces reveals that L7Ae buries more surface area (red) than YbxF and L30e and is unique in its distribution of interacting residues. The accessible surface area for YbxF was calculated subsequent to the addition of side-chain rotamers similar to L7Ae at residues K17 and K21 (α2) and E72 (α4–β4 loop).
FIGURE 4.
FIGURE 4.
Binding of YbxF to the K-turn is modulated by the stability of the canonical (C) helix. Base pairs for the B. subtilis lysC K-turn are denoted as open circles because no structure is available. Base pairs for the B. subtilis yitJ K-turn are denoted with Leontis-Westhof symbols based on PDB entry 3NPB (Lu et al. 2010). Base-pair mutations (bold) in the lysC riboswitch generate a K-turn that is capable of recognition by YbxF. Mutation of the C stem of the yitJ K-turn, similar to the wild-type sequence of lysC K-turn, results in weaker binding by YbxF. Values reported for wild-type yitJ and the single-base-pair mutant do not differ given the precision of the experiments.
FIGURE 5.
FIGURE 5.
Structure of YlxQ. (A) Cartoon representation of the overall structure of YlxQ, with secondary structure elements labeled as in Figure 1A. (B) Portion of the 1.55 Å resolution anneal-omit 2|Fo| − |Fc| electron density map corresponding to the α4–β4 loop of YlxQ (outlined in panel A) is shown contoured at 2.5σ. The corresponding α4–β4 loop from L7Ae (only main chain atoms) is shown in gray for comparison. Cα atoms are shown as enlarged spheres to highlight the main chain trajectories of YlxQ and L7Ae. (C) Structural alignment of YlxQ (blue) and YbxF (red) and (D) of YlxQ (blue) and L7Ae (gray).
FIGURE 6.
FIGURE 6.
Comparison of YlxQ with YbxF. (A) Sequence conservation among YbxF and YlxQ homologs calculated with the ConSurf server (Ashkenazy et al. 2010). (Dark red) Highly conserved among the top 65 YbxF homologs and 150 YlxQ homologs; (cyan) highly variable; (white) average conservation; (yellow) insufficient data. Select residues at equivalent positions at the RNA–protein interface on YbxF, YlxQ, and RNA are labeled. (B) Continuum electrostatic potentials at the solvent-accessible surfaces of YlxQ and YbxF. The view is from YbxF-bound RNA toward the YbxF–RNA interface. (Blue) Positive potential; (red) negative potential; (white) neutral. (C) Electrophoretic mobility shift assay showing that structure-based engineering of YlxQ fails to confer tighter K-turn binding.
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