Bioinformatics searches of eubacterial genomes have yielded many riboswitch candidates where the identity of the ligand is not immediately obvious on examination of associated genes. One of these motifs is found exclusively in the family Streptococcaceae within the 59 untranslated regions (UTRs) of genes encoding the hypothetical membrane protein classified as COG4708 or DUF988. While the function of this protein class is unproven, a riboswitch binding the queuosine biosynthetic intermediate pre-queuosine 1 (preQ 1 ) has been identified in the 59 UTR of homologous genes in many Firmicute species of bacteria outside of Streptococcaceae. Here we show that a representative of the COG4708 RNA motif from Streptococcus pneumoniae R6 also binds preQ 1 . Furthermore, representatives of this RNA have structural and molecular recognition characteristics that are distinct from those of the previously described preQ 1 riboswitch class. PreQ 1 is the second metabolite for which two or more distinct classes of natural aptamers exist, indicating that natural aptamers utilizing different structures to bind the same metabolite may be more common than is currently known. Additionally, the association of preQ 1 binding RNAs with most genes encoding proteins classified as COG4708 strongly suggests that these proteins function as transporters for preQ 1 or another queuosine biosynthetic intermediate.

Cell-cell contact–dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX−/− mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors.

[reaction: see text] Anhydrous hydrogen iodide is generated in situ by the reaction of solid iodine and a thiol. The HI thus generated has been employed for the efficient preparation of alpha-glycosyl iodides and vicinal iodohydrins from the corresponding glycosyl acetates and epoxides, respectively, and for Ferrier glycosylation of alcohols and thiols.

The modified RNA base queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) occurs in tRNA via a unique base exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. (Nature Structural Biology (2003) 10, 781-788); however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that product RNA dissociation from the enzyme is overall rate-limiting in the steady-state. Interestingly, studies comparing RNA with a 2′-deoxyriboside at the site of modification suggest a role for the 2′-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during covalent complex breakdown.Over one hundred chemically distinct modified bases are known to occur in RNA, the majority of which occur in tRNA (1). Of these, queuine (Q) stands out for several reasons. Structurally, queuine is the only modified base that is not a purine or pyrimidine analog. Instead, queuine features a pyrrolo-pyrimidine heterocyclic scaffold that is further elaborated through exocyclic chemical modifications. Perhaps most interestingly, the mechanism of incorporation of queuine into tRNA is unique. The queuine base is post-transcriptionally introduced into tRNA via a transglycosylation reaction that is catalyzed by tRNA-guanine transglycosylase (TGT) (Figure 1). Among the known modified bases, only pseudouridine is installed in an analogous manner whereby pseudouridine synthase breaks and reforms the glycosidic bond to the uracil (2).As with pseudouridine synthase, the chemical mechanism of the TGT reaction has been studied for some time. Two distinct mechanisms have been proposed and subsequently investigated for the TGT catalyzed base-exchange reaction. It was first envisaged that a dissociative mechanism, involving the intermediacy of an oxocarbonium ion, could drive the cleavage of † This work was supported in part by the National Institutes of Health (GM065489, GAG; GM07767, JDK trainee), and the University the glycosidic bond leading to the incorporation of the modified base. Alternatively, and equally plausible, the TGT reaction may proceed through a covalent TGT-RNA intermediate (3) in an associative mechanism similar to that of retaining glycosyl hydrolases (4) ( Figure 2A). In fact, biochemical studies from our laboratory have implicated the associative mechanism and the involvement of two active site aspartate residues in the reaction (5,6). Consistent with the associative mechanism, we have previously shown that the E. coli TGT follows ping-pong kinetics (7), with...

A sialyl Lewis X (sLe(x)) mimetic compound, 2-(trimethylsilyl)ethyl 3-O-carboxymethyl-beta-D-galactopyranosyl-(1-->4)-[alpha-L-fucosyl-(1-->6)]-beta-D-glucopyranoside (2a), has been synthesized in 14 steps from D-lactose. This synthesis features the use of the activated glycosylating donor, lactosyl iodide, in a Koenigs-Knorr sequence, the regioselective derivatization at the C-3 position of the galactose moiety, and the stereoselective construction of a fucose-alpha(1-->6)-lactose linkage. The mimetic was tested for its ability to inhibit human polymorphonuclear leukocyte (hPMNL) adhesion to immobilized recombinant human E-selectin under shear stress conditions.