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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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Global rearrangement of the riboswitch induced by c-di-GMP binding. (a) Low-resolution molecular envelope reconstruction based on SAXS data of the riboswitch in the presence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~90 Å. (b) Molecular envelope based on SAXS data of the riboswitch in the absence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~100 Å. The two arms of this reconstruction have dimensions approximately corresponding to those of P2 and P1b. (c) Results of nuclease probing summarized on the bound-state secondary structure. Stars denote RNase T1 protection upon c-di-GMP binding; filled ovals, RNase V1 protection upon c-di-GMP binding; open ovals, increased RNase V1 cleavage upon c-di-GMP binding. (d) Section of the autoradiogram highlighting protections observed due to their presence in the inter-helical interface, or indirectly, from stabilization of the interface between P1b and P2. (See Supplementary Fig. 10 for complete autoradiogram; this panel shows the lanes corresponding to 3.0 mM Mg2+). "+" and "−" denote conditions with 70 µM and 0 µM c-di-GMP, respectively. "NR" and "BH" denote "no reaction" and "base hydrolysis".
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Figure 4: Global rearrangement of the riboswitch induced by c-di-GMP binding. (a) Low-resolution molecular envelope reconstruction based on SAXS data of the riboswitch in the presence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~90 Å. (b) Molecular envelope based on SAXS data of the riboswitch in the absence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~100 Å. The two arms of this reconstruction have dimensions approximately corresponding to those of P2 and P1b. (c) Results of nuclease probing summarized on the bound-state secondary structure. Stars denote RNase T1 protection upon c-di-GMP binding; filled ovals, RNase V1 protection upon c-di-GMP binding; open ovals, increased RNase V1 cleavage upon c-di-GMP binding. (d) Section of the autoradiogram highlighting protections observed due to their presence in the inter-helical interface, or indirectly, from stabilization of the interface between P1b and P2. (See Supplementary Fig. 10 for complete autoradiogram; this panel shows the lanes corresponding to 3.0 mM Mg2+). "+" and "−" denote conditions with 70 µM and 0 µM c-di-GMP, respectively. "NR" and "BH" denote "no reaction" and "base hydrolysis".

Mentions: To gain further insight into the nature of the c-di-GMP-induced conformational change, we calculated low-resolution molecular envelopes16 employing SAXS data collected at physiologic Mg2+ concentration. Reconstructions based on the ligand-bound state SAXS data employing a range of starting maximum molecular dimension (Dmax) values robustly converged into an envelope with high shape complementarity (correlation coefficient = 0.94) to our cocrystal structure (with the U1A binding site replaced by a model of identical length to wild-type L2), and Dmax~90 Å (Fig. 4a and Supplementary Fig. 5, Supplementary Fig. 6a,b). Reconstructions based on the ligand-free state SAXS data converged on a more elongated molecular envelope (Dmax~100 Å) with two prominent arms (Fig. 4b and Supplementary Fig. 6c,d). The shape of this envelope is distinctly different from that of the c-di-GMP-bound state. In addition to obvious limitations due to its low resolution, interpretation of this envelope has the caveat that the ligand-free form of the RNA might explore multiple conformations in solution. However, we found that reconstructions based on ligand-free SAXS data do converge to molecular envelopes that are self-consistent at each of the three Mg2+ concentrations analyzed. Moreover, the variability of the reconstructions for any one of the three ligand-free conditions is comparable to that of the reconstructions from the ligand-bound SAXS data (Supplementary Fig. 7). We conclude that building molecular models to fit the SAXS-based molecular envelope reconstruction of the ligand-free riboswitch is justified, keeping in mind that the level of detail implied by such models is limited to the approximate location and orientation of helices.


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)

Global rearrangement of the riboswitch induced by c-di-GMP binding. (a) Low-resolution molecular envelope reconstruction based on SAXS data of the riboswitch in the presence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~90 Å. (b) Molecular envelope based on SAXS data of the riboswitch in the absence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~100 Å. The two arms of this reconstruction have dimensions approximately corresponding to those of P2 and P1b. (c) Results of nuclease probing summarized on the bound-state secondary structure. Stars denote RNase T1 protection upon c-di-GMP binding; filled ovals, RNase V1 protection upon c-di-GMP binding; open ovals, increased RNase V1 cleavage upon c-di-GMP binding. (d) Section of the autoradiogram highlighting protections observed due to their presence in the inter-helical interface, or indirectly, from stabilization of the interface between P1b and P2. (See Supplementary Fig. 10 for complete autoradiogram; this panel shows the lanes corresponding to 3.0 mM Mg2+). "+" and "−" denote conditions with 70 µM and 0 µM c-di-GMP, respectively. "NR" and "BH" denote "no reaction" and "base hydrolysis".
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Related In: Results  -  Collection

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Figure 4: Global rearrangement of the riboswitch induced by c-di-GMP binding. (a) Low-resolution molecular envelope reconstruction based on SAXS data of the riboswitch in the presence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~90 Å. (b) Molecular envelope based on SAXS data of the riboswitch in the absence of c-di-GMP (at 2.5 mM Mg2+). The maximum linear dimension of the reconstruction is ~100 Å. The two arms of this reconstruction have dimensions approximately corresponding to those of P2 and P1b. (c) Results of nuclease probing summarized on the bound-state secondary structure. Stars denote RNase T1 protection upon c-di-GMP binding; filled ovals, RNase V1 protection upon c-di-GMP binding; open ovals, increased RNase V1 cleavage upon c-di-GMP binding. (d) Section of the autoradiogram highlighting protections observed due to their presence in the inter-helical interface, or indirectly, from stabilization of the interface between P1b and P2. (See Supplementary Fig. 10 for complete autoradiogram; this panel shows the lanes corresponding to 3.0 mM Mg2+). "+" and "−" denote conditions with 70 µM and 0 µM c-di-GMP, respectively. "NR" and "BH" denote "no reaction" and "base hydrolysis".
Mentions: To gain further insight into the nature of the c-di-GMP-induced conformational change, we calculated low-resolution molecular envelopes16 employing SAXS data collected at physiologic Mg2+ concentration. Reconstructions based on the ligand-bound state SAXS data employing a range of starting maximum molecular dimension (Dmax) values robustly converged into an envelope with high shape complementarity (correlation coefficient = 0.94) to our cocrystal structure (with the U1A binding site replaced by a model of identical length to wild-type L2), and Dmax~90 Å (Fig. 4a and Supplementary Fig. 5, Supplementary Fig. 6a,b). Reconstructions based on the ligand-free state SAXS data converged on a more elongated molecular envelope (Dmax~100 Å) with two prominent arms (Fig. 4b and Supplementary Fig. 6c,d). The shape of this envelope is distinctly different from that of the c-di-GMP-bound state. In addition to obvious limitations due to its low resolution, interpretation of this envelope has the caveat that the ligand-free form of the RNA might explore multiple conformations in solution. However, we found that reconstructions based on ligand-free SAXS data do converge to molecular envelopes that are self-consistent at each of the three Mg2+ concentrations analyzed. Moreover, the variability of the reconstructions for any one of the three ligand-free conditions is comparable to that of the reconstructions from the ligand-bound SAXS data (Supplementary Fig. 7). We conclude that building molecular models to fit the SAXS-based molecular envelope reconstruction of the ligand-free riboswitch is justified, keeping in mind that the level of detail implied by such models is limited to the approximate location and orientation of helices.

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