Purine Riboswitch


by Amanda Abramson

Introduction:

In general, a riboswitch is a naturally occurring sensor that directly controls gene expression through its ability to bind various small molecule metabolites. This molecule in particular is a guanine-responsive riboswitch that controls the transcription of genes through the binding of hypoxanthine, guanine or xanthine. This control is associated with purine metabolism in multiple bacterial species because it regulates many of the operons in the purine biosynthesis pathway.

The basic structure of this riboswitch contains three intertwined helices. There are two domains in this riboswitch but only the binding domain was crystallized. The binding domain contains the binding pocket and the P1 helix . The stability of the P1 helix determines the switching domain's conformation. The guanine binding pocket is ordered upon the interaction of loop two with loop three.

Ligand Binding

When the ligand binds, the P1 helix is stabilized which orders the riboswitch core . This ordering causes the switching domain to change to the more stable terminator conformation which is shown in the top pathway in the diagram. If the ligand does not bind, the P1 helix is not stabilized and the riboswitch core remains disordered. The switching domain is then able to use part of the P1 helix to form a more stable antiterminator complex as shown in the diagram's bottom pathway. So, the P1 helix and switching domain control gene expression through their conformation because they determine if transcription is terminated or not. This control of transcription is imposed through the concentration of guanine, hypoxanthine or xanthine. At high concentrations the ligand is bound in the pocket and forms stable stacking interactions and base triplets. These prevent the incorporation of the P1 helix into the antiterminator hairpin so the terminator can form and stop transcription. On the other hand, at low concentrations of ligand the 3' side of the isolated P1 helix is able to form a stable antiterminator hairpin with the switching domain and thus, allow for continued transcription.

Conserved Nucleotides

There are conserved nucleotides in guanine riboswitches because they are useful to many different cells. In the diagram, the red nucleotides are conserved in more than 90% of known guanine riboswitches. The loop-loop core bases are highly conserved because their interaction is essential for ligand binding even though their interaction forms independently of guanine, hypoxanthine and xanthine. This is very important for the cell because the loop-loop interaction organizes the binding domain for purine recognition which leads to transcription termination. The purine binding pocket is also highly conserved around its three way junction element which forms two base triplets above and two base triplets below where the ligand binds.These regions are conserved because they are the most important parts of the riboswitch.

Loop-Loop interaction


The kissing hairpin interaction is between loops two and three . These two terminal loops are connected by hydrogen bonds between A65 with G37 and A66 with G38 . These hydrogen bonds cement the loop-loop core parallel to each other. This change in conformation orders the binding pocket so a ligand can bind. The loops are defined by two previously unobserved base quadruples. The first is made of G37, C61, U34 and A65 . The second quadruple is between G38, C60, A33 and A66 . Both quadruples consist of a Watson-Crick pair with a noncanonical pair docked into its minor grove.

Binding pocket

The natural guanine responsive riboswitch complexed with the metabolite hypoxanthine. Click here to zoom in to the hypoxanthine . The binding pocket is made of four base triplets: . The first triplet, U22, A52 and A73 of the binding pocket . The second triplet, A23, G46 and C53 of the binding pocket . The third triplet, A21, U75 and C50 of the binding pocket . The fourth triplet, U20, A76 and U49 of the binding pocket .

Hydrogen Bond Interactions

The hypoxanthine hydrogen bonds with four nucleotides U22 C74 U51 U47 to stabilize the binding pocket. It is these hydrogen bonds which form a base quadruple which stacks directly on top of the P1 helix . In the pocket all of hypoxanthine's functional groups are bound and there is space for guanine's exocyclic amino group. This shows the pocket's specificity for its ligands. Guanine's amino protons can hydrogen bond with the carbonyl oxygens of C74 and U51 which increases the binding pocket's affinity for guanine tenfold over hypoxanthine. Adenine can not bind because there would be steric hindrance between its functional groups and the pocket. However, if you mutate C74 from cytosine to uracil the binding specificity will change to favor adenine. This is because the carbonyl on hypoxanthine would change to an amino group at C6 to become adenine and the amino group of cytosine 74 at C4 changes to a carbonyl to become uracil .

Conclusion

Hypoxanthine, Guanine or Xanthine are the "keystones" for the riboswitch. This is because the binding of the ligand allows the riboswitch to direct mRNA folding along two different pathways. If the ligand binds, the P1 helix is ordered and stabilized which allows a stable terminator hairpin to form. This terminator hairpin is followed by a long sequence of U's which with the hairpin causes the RNA Pol to fall off and transcription to stop. However, if the ligand does not bind the P1 helix is not stabilized and remains disordered. As a result, part of the P1 helix is used to form a stable antiterminator hairpin. This antiterminator keeps transcription on by disrupting the terminator sequence and thus, keeping RNA Pol bound. So, this riboswitch is an effective biosensor of intracellular Guanine, Hypoxanthine and Xanthine concentrations which helps to regulate purine biosynthesis.

Bibliography

PDB Code: 1U8D

Batey, Robert, Gilbert, Sunny and Montange, Rebecca. "Structure of a natural guanine responsive riboswitch complexed with the metabolite hypoxanthine." Nature 432 (2004): 411-415.

Mandal. M. & Boese, B., Barrick, J.El, Winkler, W.C, & Breaker, R. R. "Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria." Cell 113 (2003): 577-586.

Mandal. M. & Breaker, R. R. "Gene regulation by riboswitches." Nature Rev. Mol. Cell. Biol. 5 (2004): 451-463.