Limits...
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.

Show MeSH

Related in: MedlinePlus

C-di-GMP and Mg2+ induced size and shape changes of the riboswitch, monitored by SAXS. Red indicates 2.5 mM Mg2+; black 10 mM Mg2+. Dots denote c-di-GMP-free conditions; solid lines, c-di-GMP-bound conditions ("a.u.", arbitrary units). (a) Kratky plot suggests local disorder of the ligand-free riboswitch even under high MgCl2 concentration (black dots). (b) Electron-pair probability analysis reveals compaction of the riboswitch upon binding the second messenger.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2925111&req=5

Figure 3: C-di-GMP and Mg2+ induced size and shape changes of the riboswitch, monitored by SAXS. Red indicates 2.5 mM Mg2+; black 10 mM Mg2+. Dots denote c-di-GMP-free conditions; solid lines, c-di-GMP-bound conditions ("a.u.", arbitrary units). (a) Kratky plot suggests local disorder of the ligand-free riboswitch even under high MgCl2 concentration (black dots). (b) Electron-pair probability analysis reveals compaction of the riboswitch upon binding the second messenger.

Mentions: Guinier analysis16 reveals that the radius of gyration (Rg) of the c-di-GMP riboswitch aptamer compacts in response to both c-di-GMP and Mg2+ (Supplementary Table 1). At a Mg2+ concentration of 0.5 mM (below physiologic), the Rg (~32 Å) is insensitive to saturating c-di-GMP. However, in the presence of physiologic (2.5 mM) Mg2+, the free RNA compacts (Rg=28.5 Å), and undergoes a further c-di-GMP-dependent compaction (Rg=23.9 Å). This latter Rg is the same, within experimental precision, as that calculated with a model of the riboswitch based on our crystal structure (with a wild-type length L2 modeled in; Supplementary Table 2 and Supplementary Fig. 5). At highly stabilizing Mg2+ (10 mM), the RNA compacts even in the absence of c-di-GMP to a Rg that is comparable to that of its c-di-GMP-bound form at physiologic Mg2+ (Supplementary Table 1). However, Kratky analysis16 (Fig. 3a) shows that even at 10 mM Mg2+, the free RNA exhibits behavior at high q indicative of local disorder, suggesting that the c-di-GMP-binding pocket and/or P1a remain unfolded in the absence of ligand. P(r) analysis16 (Fig. 3b) reveals a marked ligand-induced compaction of the RNA at physiologic Mg2+, and also a modest c-di-GMP-induced conformational change at 10 mM Mg2+, likely indicative of folding of the second messenger-binding pocket.


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)

C-di-GMP and Mg2+ induced size and shape changes of the riboswitch, monitored by SAXS. Red indicates 2.5 mM Mg2+; black 10 mM Mg2+. Dots denote c-di-GMP-free conditions; solid lines, c-di-GMP-bound conditions ("a.u.", arbitrary units). (a) Kratky plot suggests local disorder of the ligand-free riboswitch even under high MgCl2 concentration (black dots). (b) Electron-pair probability analysis reveals compaction of the riboswitch upon binding the second messenger.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925111&req=5

Figure 3: C-di-GMP and Mg2+ induced size and shape changes of the riboswitch, monitored by SAXS. Red indicates 2.5 mM Mg2+; black 10 mM Mg2+. Dots denote c-di-GMP-free conditions; solid lines, c-di-GMP-bound conditions ("a.u.", arbitrary units). (a) Kratky plot suggests local disorder of the ligand-free riboswitch even under high MgCl2 concentration (black dots). (b) Electron-pair probability analysis reveals compaction of the riboswitch upon binding the second messenger.
Mentions: Guinier analysis16 reveals that the radius of gyration (Rg) of the c-di-GMP riboswitch aptamer compacts in response to both c-di-GMP and Mg2+ (Supplementary Table 1). At a Mg2+ concentration of 0.5 mM (below physiologic), the Rg (~32 Å) is insensitive to saturating c-di-GMP. However, in the presence of physiologic (2.5 mM) Mg2+, the free RNA compacts (Rg=28.5 Å), and undergoes a further c-di-GMP-dependent compaction (Rg=23.9 Å). This latter Rg is the same, within experimental precision, as that calculated with a model of the riboswitch based on our crystal structure (with a wild-type length L2 modeled in; Supplementary Table 2 and Supplementary Fig. 5). At highly stabilizing Mg2+ (10 mM), the RNA compacts even in the absence of c-di-GMP to a Rg that is comparable to that of its c-di-GMP-bound form at physiologic Mg2+ (Supplementary Table 1). However, Kratky analysis16 (Fig. 3a) shows that even at 10 mM Mg2+, the free RNA exhibits behavior at high q indicative of local disorder, suggesting that the c-di-GMP-binding pocket and/or P1a remain unfolded in the absence of ligand. P(r) analysis16 (Fig. 3b) reveals a marked ligand-induced compaction of the RNA at physiologic Mg2+, and also a modest c-di-GMP-induced conformational change at 10 mM Mg2+, likely indicative of folding of the second messenger-binding pocket.

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