Abstract
We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA.
Highlights
Riboswitches are cis-acting regulatory elements often found in the 5′-untranslated regions (UTR) of bacterial mRNA [1,2,3]
The structure suggests how the riboswitch could be divided into two separate RNA molecules, and we show that addition of L-glutamine induces the formation of a functional ligand binding domain
The structure was determined by molecular replacement using PHASER [18] and a search model derived by Rosetta homology modeling using the stepwise Monte-Carlo algorithm [24]
Summary
Riboswitches are cis-acting regulatory elements often found in the 5′-untranslated regions (UTR) of bacterial mRNA [1,2,3] They usually bind cellular metabolites resulting in up- or down-regulation of the downstream gene at the transcriptional or translational level. Regulation of nitrogen assimilation [4,5] is important in marine bacteria such as the photosynthetic cyanobacteria, where the key enzyme is glutamine synthetase (GS) that condenses glutamate with ammonia to form glutamine [6] This is subject to several forms of regulation, including the transcriptional regulator NtcA and the GS-inactivating protein factors IF7 and IF17, encoded by gifA and gifB, respectively [7,8]. Given the proximity to the initiator codon (Supplementary Figure S1), it is likely to act as a translational riboswitch
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