title: Analysis of mRNA recognition by human thymidylate synthase.
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.
authors.affiliation: *Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, U.S.A.
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Brunn
authors.affiliation: *Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, U.S.A.
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Dibrov
authors.affiliation: *Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, U.S.A.
authors.forename: Melody B
authors.initials: MB
authors.lastname: Kao
authors.affiliation: *Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, U.S.A.
authors.forename: Majid
authors.initials: M
authors.lastname: Ghassemian
authors.affiliation: *Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, U.S.A.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hermann
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018387:Codon, Initiator / Q000235:genetics; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D018387:Codon, Initiator; D012333:RNA, Messenger; D013940:Thymidylate Synthase