James W. Bodley

Studies on translocation. 21. Steady state kinetic analysis of the mechanism of guanosine triphosphate hydrolysis catalyzed by Escherichia coli elongation factor G and the ribosome

The mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor G and the ribosome in the absence of other participants in protein synthesis was examined by steady-state kinetic analysis. Optimal hydrolytic conditions were determined to be approximately pH 8.0, 20 mM Mg2+, and 80 mM NH4+. Kinetic analyses were performed under these conditions at constant elongation factor G concentrations and variable ribosome and GTP concentrations. The resulting double-reciprocal plots in conjunction with the inhibition patterns obtained with GDP indicated that the reaction occurs by an ordered mechanism in which GTP is the leading obligatory substrate. Dissociation constants for GTP and guanosine diphosphate (GDP), as well as limiting Michaelis constants for GTP and ribosomes, were calculated from the double-reciprocal plots. These values are: KSGTP = 37.0 muM, KSGDP = 16.5 muKMGTP = 8.0 muM, KMR = 0.22 muM. Inhibition was also observed at high ribosomal concentrations and suggests that inhibition was due both to the decreased breakdown of the tertiary elongation factor G-GDP-ribosome posthydrolytic complex and to the formation of a nonproductive elongation factor G-ribosome complex. A sequential mechanism with a dead-end elongation factor G-ribosome complex has been constructed to describe the hydrolysis of GTP catalyzed by elongation factor G and the ribosome.

[38] Diphthamide in elongation factor 2: ADP-ribosylation, purification, and properties

Publisher Summary This chapter describes ADP-ribosylation, purification, and properties of diphthamide in elongation factor 2 (EF-2). Diphthamide is a posttranslational derivative of histidine which occurs in a single location in protein synthesis EF-2. The occurrence of diphthamide was first found through the study of a second post translational modification reaction, the ADP-ribosylation of EF-2 by diphtheria toxin. The purification and study of EF-2 and diphthamide revolve around the specificity and nature of the diphtheria toxin reaction. With the aid of toxin and radioactive NAD+ (either [adenine-2,8-3H]NAD+ or [32P]NAD+) it is possible to specifically radiolabel EF-2 either in crude extracts or in purified protein preparations. The reaction is specific in that only a single eukaryotic polypeptide is labeled and there are no toxinspecific ADP-ribose acceptors in eubacterial extracts. The toxin reaction also provides a direct measure of the quantity of EF-2 because the reaction is both irreversible and stoichiometric. The preparation of large quantities of ADP-ribosyl EF-2 is facilitated by the partial purification of the protein prior to its modification by diphtheria toxin. When performed on a small scale, a single chromatography step is sufficient to purify the protein approximately 10- to 15-fold and to remove interfering substances so that EF-2 can be ADP-ribosylated without dilution.

[24] The binding of Escherichia coli elongation factor G to the ribosome

Publisher Summary Elongation factor G (EF G) is one of the soluble factors involved in the process of peptide chain elongation. It is required for translocation to occur, i.e., the movement of the ribosome relative to the mRNA. GTP is required for EF G to function in the overall reaction, and GTP is hydrolyzed to GDP and Pi. In addition, EF G in the presence of ribosomes, but in the absence of other components of protein synthesis, catalyzes the hydrolysis of GTP. Both the EF G-dependent translocation and the “uncoupled” GTP hydrolysis are inhibited by the steroid antibiotic fusidic acid? Recent studies have shown that EF G binds to the ribosome in a reaction dependent on either GTP or GDP to form a labile ribosome·EF G·GDP complex.

On the interaction of periodate oxidized GDP and its borohydride reduction product with the elongation factors Tu and G from Escherichia coli

Nothing is known about the nucleotide binding sites of polypeptide chain elongation factor EF-Tu or on the EF-G-ribosome complex (nomenclature as in ref. [ 11). Also, so far no difference has been reported between their substrate specificities (reviewed in ref. [2]). We have been investigating derivatives of GTP and GDP as an attack on both of these questions. In order to form a reactive derivative of GTP or GSP which may be useful as an active site label, we followed the strategy of Erlanger and Beiser [3]. They made an immunogenic adduct between nucleotides and serum albumin. First, they formed the dialdehyde derivative by periodated oxidation. Then they made the Schiff s base between this and the amino groups of the protein. Finally, they made a stable covalent link by borohydride reduction. With this strategy in mind, we prepared periodate oxidized GTP (GTPoX) and its borohydride reduction product (GTPoX-‘Cd). We have found that both can substitute for GDP in the binding site of the EF-G- ribosome complex, but neither can bind to EF-Tu. While we have thus far been unable to form a covalent link between GDPoX and the guanine nucleotide bind- ing site, the experiments revealed an important differ- ence between the substrate specificities of EF-Tu and EF-G. 2.

EFFECT OF ANTIBIOTICS AND RIBONUCLEASE ON POLYPEPTIDE AND PROTEIN BIOSYNTHESIS IN DIFFERENT STRAINS OF BACILLUS BREVIS.

The effect of chloramphenicol, puromycin, and ribonuclease (EC 2.7.7.16) on polypeptide and protein biosynthesis was tested in growing cultures and in cell-free systems of three different strains of Bacillus brevis. With all three strains of the organism, the peptide-synthesizing ability was completely or severely inhibited under conditions which caused total or marked blockage of protein formation. The results support our previous conclusion, based on studies with ribosomal and soluble cellular components, that gramicidins, tyrocidines, and gramicidin S molecules are all synthesized by a pathway which resembles that of protein biogenesis.

[11] The measurement of cyclic GMP with Escherichia coli elongation factor tu

Publisher Summary Elongation factor Tu (EF-Tu) is a soluble factor from E. coli that promotes the binding of aminoacyl-tRNA to ribosomes in the process of peptide chain elongation. EF-Tu is easily obtainable in a reasonably good state of purity, is relatively stable, exhibits a high degree of specificity for GDP (and GTP) and a dissociation constant for GDP in the range of 10 -9 M (10 -7 M for GTP). Highly purified EF-Tu can be prepared from Escherichia coli (B cells obtainable from Grain Processing Corporation, Muscatine, Iowa) by the method of Miller and Weissbach or Arai. The procedure involves preparation of a high speed supernatant fraction from broken cells followed by partial purification of an EF-Tu-GDP complex by ammonium sulfate fractionation, chromatography on DEAE Sephadex A-50, and Sephadex G-100. The specificity of the assay derives from three steps in the procedure: (a) the chromatographic purification of tissue extracts, (b) the selective conversion of GMP catalyzed by ATP-GMP phosphotransferase, and (c) the selective binding by EF-Tu of GDP (and GTP).