Download Purine Riboswitch

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 guanineresponsive 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.