Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these mutants cannot carry out chain elongation. To define more precisely the function of base 530, we have studied ribosomes in which G530 was mutated to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits and could combine with 50S to make 70S ribosomes. 16S rRNA in vitro transcripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) synthesis, P- and A-site tRNA binding, and 30S initiation complex formation. 30S initiation complexes, in the presence of 50S, could react with puromycin like the wild-type. The rate, extent, and position of cross-linking of AcVal-tRNA in the P site to 16S RNA were identical in mutant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe-tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defective in carrying out synthesis of fMet-Val dipeptide using natural mRNA. Mutant 30S ribosomes were also refractory to streptomycin-induced misreading although no misreading was observed in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containin? om'i ~"~m c H-ras were analyzed by 500-MHz 'H-NMR spectroscopy and binding to immobilized lectins. The cells were mc1;iholically labeled with ~-[~H]glucosamine or ~- [~H]fucose and the glycopeptides included in Bio-Gel P-10 ( M , 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues.The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 'H-NMR spectroscopy to be tetraantennary containing terminal Galal -+ 3.This structure was verified by binding to the immobilized a-Gal-specific lectin, Grijfonia simplicijolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri-or tetraantennary glycopeptides.In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAca2+3 in addition to Galal+3. Fucal-thIn attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAca2 + 3 reduced the affinity to L-PHA -agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAca2+3 but no NeuNAca2-+6 or Galal + 3 .A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAca2+3, the presence of NeuNAca2-6 and Galal +3.On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri-or tetraantennae must be sialylated in a2-3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in aGal residues, whereas after transformation a-Gal residues appear to be replaced by NeuNAca2+ 3. Thus, in this case, transformation may be accompanied by a change in the glycosyltransferases responsible for termination of the branches rather than those conferring branch configurations.

The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.

No abstract