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Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch.

Kulshina N, Baird NJ, Ferré-D'Amaré AR - Nat. Struct. Mol. Biol. (2009)

Bottom Line: The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation.These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger.Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA.

ABSTRACT
The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

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Overall structure of the c-di-GMP riboswitch. (a) Unbiased /Fo/-/Fc/ electron density corresponding to the bound c-di-GMP before it was included in the crystallographic model, superimposed on the refined model of the ligand (map contoured at 3.0 s.d.) (b) Portion of a composite simulated annealing-omit 2/Fo/-/Fc/ Fourier synthesis42 calculated with the final crystallographic model, contoured around A82 at 1.5 s.d. (c) Schematic representation of the structure of the riboswitch bound to c-di-GMP. Thin black lines with arrowheads depict connectivity, dashed green lines, long-range stacking interactions, and the red line the inter-helical base pair. Leontis-Westhof symbols43 depict non-canonical base pairs. Except for the U1A binding site, the numbering scheme is that of ref. 6, which is used throughout. (d) Cartoon representation of the three-dimensional structure. Color coding as in (c). The U1A-RBD is shown as a gray ribbon.
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Figure 1: Overall structure of the c-di-GMP riboswitch. (a) Unbiased /Fo/-/Fc/ electron density corresponding to the bound c-di-GMP before it was included in the crystallographic model, superimposed on the refined model of the ligand (map contoured at 3.0 s.d.) (b) Portion of a composite simulated annealing-omit 2/Fo/-/Fc/ Fourier synthesis42 calculated with the final crystallographic model, contoured around A82 at 1.5 s.d. (c) Schematic representation of the structure of the riboswitch bound to c-di-GMP. Thin black lines with arrowheads depict connectivity, dashed green lines, long-range stacking interactions, and the red line the inter-helical base pair. Leontis-Westhof symbols43 depict non-canonical base pairs. Except for the U1A binding site, the numbering scheme is that of ref. 6, which is used throughout. (d) Cartoon representation of the three-dimensional structure. Color coding as in (c). The U1A-RBD is shown as a gray ribbon.

Mentions: Previous analyses of the V. cholerae tfoX c-di-GMP riboswitch6 delineated the minimal 5'- and 3'- RNA boundaries needed for tight binding (Kd≤10 nM) to the second messenger (the 'aptamer domain'). A phylogenetically conserved helix (paired region P2) is closed by a loop (L2) that is highly variable5. Our crystallization construct encompasses these boundaries, and incorporates8 a binding site for the spliceosomal protein U1A in place of L2. Consistent with lack of conservation of L2, this construct binds both c-di-GMP and the U1A RNA-binding domain (RBD) (Supplementary Fig. 1). We solved the structure de novo by molecular replacement employing the U1A-RBD cognate complex9 and short A-form duplexes of arbitrary sequence as search models, analogous to what was reported for two ribozymes10,11 and an unrelated riboswitch12. This procedure13 unambiguously revealed the bound c-di-GMP in residual electron density maps phased with models that had never included the second messenger (Fig. 1a), and allowed complete tracing of both RNA molecules in the asymmetric unit (Fig. 1b). The crystallographic model (Rfree=29.2% at 3.2 Å resolution) has a mean coordinate precision of 0.5 Å (Methods and Table 1).


Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch.

Kulshina N, Baird NJ, Ferré-D'Amaré AR - Nat. Struct. Mol. Biol. (2009)

Overall structure of the c-di-GMP riboswitch. (a) Unbiased /Fo/-/Fc/ electron density corresponding to the bound c-di-GMP before it was included in the crystallographic model, superimposed on the refined model of the ligand (map contoured at 3.0 s.d.) (b) Portion of a composite simulated annealing-omit 2/Fo/-/Fc/ Fourier synthesis42 calculated with the final crystallographic model, contoured around A82 at 1.5 s.d. (c) Schematic representation of the structure of the riboswitch bound to c-di-GMP. Thin black lines with arrowheads depict connectivity, dashed green lines, long-range stacking interactions, and the red line the inter-helical base pair. Leontis-Westhof symbols43 depict non-canonical base pairs. Except for the U1A binding site, the numbering scheme is that of ref. 6, which is used throughout. (d) Cartoon representation of the three-dimensional structure. Color coding as in (c). The U1A-RBD is shown as a gray ribbon.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925111&req=5

Figure 1: Overall structure of the c-di-GMP riboswitch. (a) Unbiased /Fo/-/Fc/ electron density corresponding to the bound c-di-GMP before it was included in the crystallographic model, superimposed on the refined model of the ligand (map contoured at 3.0 s.d.) (b) Portion of a composite simulated annealing-omit 2/Fo/-/Fc/ Fourier synthesis42 calculated with the final crystallographic model, contoured around A82 at 1.5 s.d. (c) Schematic representation of the structure of the riboswitch bound to c-di-GMP. Thin black lines with arrowheads depict connectivity, dashed green lines, long-range stacking interactions, and the red line the inter-helical base pair. Leontis-Westhof symbols43 depict non-canonical base pairs. Except for the U1A binding site, the numbering scheme is that of ref. 6, which is used throughout. (d) Cartoon representation of the three-dimensional structure. Color coding as in (c). The U1A-RBD is shown as a gray ribbon.
Mentions: Previous analyses of the V. cholerae tfoX c-di-GMP riboswitch6 delineated the minimal 5'- and 3'- RNA boundaries needed for tight binding (Kd≤10 nM) to the second messenger (the 'aptamer domain'). A phylogenetically conserved helix (paired region P2) is closed by a loop (L2) that is highly variable5. Our crystallization construct encompasses these boundaries, and incorporates8 a binding site for the spliceosomal protein U1A in place of L2. Consistent with lack of conservation of L2, this construct binds both c-di-GMP and the U1A RNA-binding domain (RBD) (Supplementary Fig. 1). We solved the structure de novo by molecular replacement employing the U1A-RBD cognate complex9 and short A-form duplexes of arbitrary sequence as search models, analogous to what was reported for two ribozymes10,11 and an unrelated riboswitch12. This procedure13 unambiguously revealed the bound c-di-GMP in residual electron density maps phased with models that had never included the second messenger (Fig. 1a), and allowed complete tracing of both RNA molecules in the asymmetric unit (Fig. 1b). The crystallographic model (Rfree=29.2% at 3.2 Å resolution) has a mean coordinate precision of 0.5 Å (Methods and Table 1).

Bottom Line: The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation.These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger.Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, Washington, USA.

ABSTRACT
The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

Show MeSH
Related in: MedlinePlus