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Biomedical subjects

B F Clark

Publications and source records attributed to B F Clark.

At least 109 records · Page 6Linked to original sources
PubMed1986

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli.

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.

Autoradiography↗
PubMed1985

Different conformations of tRNA in the ribosomal P-site and A-site.

Footprinting studies involving radioactively end-labelled tRNA species bound at either the ribosomal P- or A-site have yielded information that the tRNA's conformation is different in the two sites. Appropriate controls showed the relevance of using poly(U)-directed tRNAPhe binding in the P-site and Phe-tRNAPhe in the A-site. Digestion of the tRNA species was effected by RNases T1, T2 and cobra venom RNase. Experiments were performed with tRNAs 32P-labelled at either end to establish positions of primary cuts more confidently. In addition to the common protection of the aminoacyl-stem and anticodon-arm, footprinting experiments revealed striking differences in the accessibility of the T- and D-loops of tRNAs bound in the P- and A-sites. We observed a more open structure for the tRNA in the A-site. These results are consistent with a dynamic structure of tRNA during the translocation step of protein biosynthesis.

Binding Sites↗
PubMed1985

A model for the tertiary structure of p21, the product of the ras oncogene.

A model was developed for the structure of p21, the protein with a molecular weight of 21,000 that is produced by the ras genes. This model predicts that p21 consists of a central core of beta-sheet structure, connected by loops and alpha helices. Four of these loops comprise the guanine nucleotide binding site. The phosphoryl binding region is made up of amino acid sequences from 10 to 16 and from 57 to 63 of p21. The latter sequence may contain a site for magnesium binding. Amino acids defining guanine specificity are Asn-116 and Asp-119, and sequences around amino acid 145 may contribute to guanine binding. The model makes it possible to visualize how oncogenic mutations of p21 affect interaction with guanine nucleotides.

Amino Acids↗
PubMed1985

Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X-ray crystallographic studies, are reported. The protein elements that take part in the nucleotide binding are located in four loops connecting beta-strands with alpha-helices. These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Escherichia coli↗
PubMed1985

Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids.

The mouse uterine epithelium under various hormonal regimes is a good system to identify biochemical events associated with cell growth, DNA synthesis and cell division. This is because estradiol-17 beta stimulates the cells to undergo a synchronized wave of DNA synthesis and cell division. Estriol, on the other hand, also stimulates DNA synthesis but because of the rapid loss of this hormone from the tissue some of the cells abort, giving a constant epithelial cell number. Three days of progesterone pretreatment, however, completely suppresses the estradiol-17 beta-induced wave of DNA synthesis and cell proliferation. Using these hormonal treatments we have shown that both estradiol-17 beta and estriol stimulate protein and rRNA synthesis with the concomitant increase of protein and rRNA per mg of DNA. These macromolecules accumulated in direct proportion to the fraction of cell committed to DNA synthesis. Estriol, however, did not sustain the growth responses and at the peak of DNA synthesis both rRNA and protein synthesis had returned to control levels. Progesterone pretreatment, despite inhibiting the proliferative response, failed to inhibit any of the estradiol-17 beta-induced increases in protein and rRNA synthesis. Indeed 12 h after estradiol-17 beta injection the cells had identical protein and rRNA contents, regardless of whether they had been exposed to progesterone or not. The present data therefore suggests that in the uterine epithelium cell growth as defined by protein and rRNA accumulation and DNA synthesis represents two independently regulated pathways.

Animals↗
PubMed1984

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

Amino Acids↗
PubMed1984

Cross-linking of tRNA at two different sites of the elongation factor Tu.

Recently, we reported on the induction by kirromycin of two tRNA binding sites on elongation factor Tu. To obtain independent information on the existence of these two sites and to characterize them further, 3' oxidized tRNA was cross-linked to elongation factor Tu by [3H]borohydride reduction. Specific cross-linking occurred exclusively in the presence of kirromycin. In the case of elongation factor Tu X GDP X kirromycin, cross-linking was found at lysine-208; in elongation factor Tu X GTP X kirromycin, cross-linking was at lysine-208 and lysine-237. In both elongation factor Tu complexes, kirromycin itself was found cross-linked to lysine-357. The tRNA cross-linking sites are in agreement with the idea of two different binding sites of tRNA on elongation factor Tu.

Anti-Bacterial Agents↗
PubMed1983

Methylation of elongation factor 1 alpha in mouse 3T3B and 3T3B/SV40 cells.

Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl-3H]methionine in the presence of cycloheximide have revealed that the elongation factor 1 alpha (EF-1 alpha) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.

Animals↗
PubMed1982

N-Tosyl-L-phenylalanylchloromethane reacts with cysteine 81 in the molecule of elongation factor Tu from Escherichia coli.

Elongation factor EF-Tu from Escherichia coli was labelled with N-[14C]tosyl-L-phenylalanylchloromethane, digested with trypsin and the peptides obtained separated by HPLC. The only radioactive peak recovered corresponded to tryptic peptide containing residues 75-98. Sequencing of the peptide by automated Edman degradation identified cysteine 81 as the site of N-tosyl-L-phenylalanylchloromethane modification. These results confirm the importance of this residue for the interaction with aminoacyl-tRNAs.

Amino Acid Chloromethyl Ketones↗
PubMed1982

The site of interaction of aminoacyl-tRNA with elongation factor Tu.

We have used RNases T1, T2 and A to digest two aminoacyl-tRNAs, Escherichia coli Phe-tRNAPhe and E. coli Met- tRNAMetm both in the naked forms and in ternary complexes with E. coli elongation factor Tu (EF-Tu) and GTP. An analysis of the 'footprinting' results has led to an interpretation that has localized the part of the three-dimensional structure of aminoacyl-tRNA covered by the protein in the ternary complex. In terms of the three-dimensional structure of tRNA established for yeast tRNAPhe, EF-Tu covers the aa-end, aa-stem, T-stem, and extra loop on the side of the L-shaped tRNA that exposes the extra loop.

Base Sequence↗
PubMed1981

The influence of elongation-factor-Tu . GTP and anticodon-anticodon interactions on the anticodon loop conformation of yeast tRNATyr.

The interactions of yeast tRNATyr, spin-labelled at position i6A-37 next to the anticodon, with EF-Tu . GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon-anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.

Anticodon↗