Abstract
Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2′-deoxyuridine-5′-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix–loop–helix domain on the protein surface was identified as the putative RNA-binding site.
Highlights
Cellular DNA synthesis critically depends on the supply of nucleotide triphosphate building blocks
We have used a combination of X-ray crystallography, in vitro translation functional studies and UV cross-linking to investigate the molecular recognition of the TS site 1 RNA motif by the enzyme
The human thymidylate synthase (hTS) structure determined at low salt (PDB: 4H1I) represents the first time that the ligand-free protein has been captured in the active conformation, which was previously observed only in a mutant containing the stabilizing mutation R163K (PDB: 2RD8) [29] and in substrate complexes, including a ternary complex of the enzyme with 2 -deoxyuridine-5 -monophosphate (dUMP) as well as the folic acid analogue inhibitor raltitrexed (PDB: 1HVY) [30]
Summary
Cellular DNA synthesis critically depends on the supply of nucleotide triphosphate building blocks. The hTS structure determined at low salt (PDB: 4H1I) represents the first time that the ligand-free protein has been captured in the active conformation, which was previously observed only in a mutant containing the stabilizing mutation R163K (PDB: 2RD8) [29] and in substrate complexes, including a ternary complex of the enzyme with dUMP as well as the folic acid analogue inhibitor raltitrexed (PDB: 1HVY) [30].
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