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J Modolell

Publications and source records attributed to J Modolell.

At least 73 records · Page 4Linked to original sources
PubMed1976

Polypeptide-chain elongation promoted by guanyl-5'-yl imidodiphosphate.

In a purified system from Escherichia coli containing ribosomes complexed with poly(uridylic acid) and N-acetyl-phenylalanyl-tRNA, the nonhydrolyzable analog of GTP, guanyl-5'-yl imidodiphosphate (Guo-5'-P2-NH-P), promotes polypeptide synthesis at a rate several times slower than GTP. The activity is completely dependent on elongation factors EF-T (i.e, EF-Ts + EF-Tu) and EF-G. Examination of individual steps of the elongation cycle in partial reactions shows that Guo-5'-P2-NH-P is as efficient as GTP in promoting the EF-T-dependent binding of phenylalanyl-tRNA to the ribosomal A site. In contrast, Guo-5'-P2-NH-P promotes the translocation-dependent binding of phenylalanyl-tRNA to a ribosome complexed with A-site-bound N-acetyl-phenylalanyl-tRNA much more slowly than GTP. This slow rate of binding is due to the presence of EF-G on the ribosome, and not to sluggish translocation, since (a) the rate remains slow even after translocation of N-acetylphenylalanyl-tRNA is completed, (b) it is greatly speeded up by removal of EF-G from the reaction mixture (after translocation has occurred), and (c) it is slowed down again by readdition of the factor. Moreover, with post-translocated ribosomes and in the absence of EF-G, formation of dipeptide subsequent to the EF-T-dependent binding of phenylalanyl-tRNA is much slower when binding of this substrate has been promoted by Guo-5'-P2-NH-P than it is when promoted by GTP. The results suggest that, during polymerization with Guo-5'-P2-NH-P, EF-G and EF-Tu are slowly released from the ribosome and, consequently, the steps of the elongation cycle subsequent to translocation and aminoacyl-tRNA binding (aminoacyl-tRNA binding and peptide bond formation, respectively) are delayed. Thus, durong elongation cycle, GTP hydrolysis is probably essential for fast release of the factors from the ribosome.

Binding Sites↗
PubMed1976

Stoichiometry of polypeptide chain elongation.

To quantitate the amount of GTP hydrolyzed during polypeptide chain elongation, an in vitro system containing purified endogenous Escherichia coli polysomes has been developed. The polysomes are washed with 1 M NH4Cl to eliminate endogenous GTPase activities and are depleted of subunits and free ribosomes to diminish the uncoupled elongation factor G-dependent GTP hydrolysis. These polysomes, supplemented with elongation factors, aminoacyl-tRNA, and low concentrations of GTP, incorporate amino acids in their nascent peptide chains. After correcting for a background of uncoupled GTP hydrolysis, it has been found that the incorporation of each molecule of amino acid is associated with the hydrolysis of 2 molecules of GTP.

Binding Sites↗
PubMed1976

A form of elongation factor G insensitive to N-ethyl-maleimide.

Treatment of elongation factor G (EF-G) with the thiol reagent N-ethylmaleimide only partially inhibits (10 to 70%) the activity of the factor in (a) guanosine nucleotide-EF-G-ribosome complex formation, (b) uncoupled ribosome-dependent GTP hydrolysis, and (c) polypeptide synthesis. Moreover, a similar treatment of the factor with N-[3H]ethylmaleimide does not lead to 3H-label being associated with a GDP-EF-G-ribosome-fusidic acid complex. Thus, the results indicate the presence in EF-G preparations of a form of the factor that does not react with N-ethylmaleimide.

Binding Sites↗
PubMed1975

The interaction of fusidic acid with peptidyl-transfer-ribonucleic-acid - ribosome complexes.

The inhibitory action of fusidic acid on peptide-chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF-G-plus-ribosome-dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl-tRNA or peptidyl-tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide-chain elongation. The role of the GDP-EF-G-ribosome-fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide-chain elongation. Using purified elongation factors the complex formed on only 25-35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, gamma-32P]GTP. In contrast, with crude factors (S 100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl-tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF-G when the donor site is occupied by peptidyl-tRNA. In the polyuridylic-acid-directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent.

Binding Sites↗
PubMed1973

The interaction of elongation factor G with N-acetylphenylalanyl transfer RNA-ribosome complexes.

N-Acetyl-Phe-tRNA, nonenzymically bound to the acceptor site of Escherichia coli ribosomes, readily undergoes translocation in the presence of elongation factor (EF)-G and GTP. The translocated N-acetyl-Phe-tRNA, bound to the ribosomal donor site, prevents further interaction of EF-G with the ribosome, for it inhibits the GTP hydrolysis that takes place in the presence of EF-G and ribosomes and it decreases the formation of either the GDP.EF-G.fusidic acid.ribosome complex or the 5'-guanylylmethylenediphosphonate.EF-G.ribosome complex. Deacylation with puromycin of the donor site-bound N-acetyl-Phe-tRNA reverses these inhibitions, even though the tRNA(Phe) moiety remains bound to the ribosme. These results suggest that ribosomes complexed with messenger RNA and peptidyl-tRNA may be restricted in their ability to interact with EF-G to that part of the elongation cycle when peptidyl-tRNA is in the acceptor site, and deacylated tRNA in the donor site. Deacylation of the donor site-bound peptidyl-tRNA associated with peptide bond formation may control the interaction of EF-G with the ribosome.

Binding Sites↗
PubMed1972

Inhibition by elongation factor EF G of aminoacyl-tRNA binding to ribosomes.

Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg(++) concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site.

Amino Acids↗
PubMed1971

Inhibition by siomycin and thiostrepton of both aminoacyl-tRNA and factor G binding to ribosomes.

Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor T(u). A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-T(u)-GTP complex and that of factor G.

Anti-Bacterial Agents↗