The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of "36 nt is protected from RNase Ti digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate to thymidine monophosphate (dTMP) and dihydrofolate (1). Because this enzymatic reaction provides the sole de novo intracellular source of dTMP, TS is a critical therapeutic target enzyme in cancer chemotherapy (2, 3).In mammalian cells, the study of TS gene regulation has mainly focused on cell cycle-directed events. The increase in TS enzyme expression that accompanies entry into the S phase of the cell cycle appears to be regulated at both the transcriptional and the posttranscriptional level (4-9). More recently, Jolliff et al. (10) protein end product, TS, in an autoregulatory manner (13). Using an in vitro rabbit reticulocyte lysate system, we observed that recombinant human TS inhibited translation of TS mRNA. Preliminary studies using a gel mobility-shift assay confirmed binding of recombinant human TS to its corresponding mRNA.Here we provide evidence that TS protein binds specifically to TS RNA sequences in vitro. Two binding sites for TS have been identified, one located within the initial 188 nt of the TS RNA and the other between nt 434 and 634. Further studies reveal that a 35-nt RNA oligomer containing the TS translational initiation sequence retains the ability to specifically bind TS. MATERIALS AND METHODS Preparation of Plasmid Constructs and in Vitro mRNATranscripts. Full-length TS RNA transcript was synthesized with SP6 RNA polymerase (13). Full-length antisense TS RNA transcript was synthesized with T7 RNA polymerase after linearization of pcEHTS with Sst I. The pcEHTS-PST RNA (PST) transcript was synthesized after linearization of pcEHTS with Pst I. The 113-bp Pst I-EcoRI fragment from pcEHTS was cloned into the Pst I and EcoRI sites of pGEM-4Z (Promega), and pcEHTS-PST/EcoRI (PSTEcoRI) RNA was synthesized with SP6 RNA polymerase after linearization with HindIII. The 133-bp EcoRI-Ava I fragment from pcEHTS was cloned into the EcoRI...