[14C]Lysine peptides synthesized in an in vitro Escherichia coli system in the presence of chloramphenicol

Julian, Gordon R.

doi:10.1016/s0022-2836(65)80277-7

Cited by 55 publications

(11 citation statements)

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“…2). When present throughout the preincubation period, nalidixic acid, a specific inhibitor of DNA replication (9), rifampin, a specific inhibitor of initiation of RNA synthesis (38), and chloramphenicol, a specific inhibitor of peptide bond formation in protein synthesis (1,14), all prevented the development of in vitro DNA synthetic ability per cell (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli

Smith¹,

Boerner²

1975

J Bacteriol

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In vitro deoxyribonucleic acid (DNA) synthesis systems based on an earlier system using penicillin have been developed which use osmotic lysis of lysozyme-formed spheroplasts of Escherichia coli cells embedded in an agarose matrix. An adenosine 5'-triphosphate (ATP)-dependent semiconservative mode, or replicative mode, of in vitro DNA synthesis is exhibited which is sensitive to nalidixic acid. These systems require growth of the agar-embedded cells in a preincubation medium before spheroplast formation and osmotic lysis. Inhibitor studies suggest that one or more required macromolecular species are synthesized during this preincubation growth period. Osmotic shock fluid from E. coli contains macromolecular factors which preferentially stimulate the ATP-dependent semiconservative mode of in vitro DNA synthesis. In some cases, the ATPindependent mode of synthesis is inhibited by shock fluid. Evidence is presented that the stimulating factors found in the osmotic shock fluid come from the E. coli periplasmic space. This stimulation is observed using either toluene-treated cells or lysed agar-embedded ethylene glycol-bis-(beta-aminoethyl ether) N,N'tetraacetate-lysozyme spheroplasts, and is thus independent of the in vitro DNA synthesis system used. Shock fluid obtained from a given E. coli dna mutant does not stimulate in vitro DNA synthesis by that mutant. However, in some cases, shock fluid from one class of dna mutants does stimulate ATP-dependent in vitro DNA synthesis by another class of dna mutants, in a thermosensitive reaction. Gently prepared cell extracts also stimulate ATP-dependent in vitro DNA synthesis, whereas cell extracts prepared by more severe procedures inhibit this in vitro synthesis. Several stimulating DNA replication factors may be present in the osmotic shock fluid, including products of E. coli dna genes.Characteristics of an in vitro deoxyribonucleic acid (DNA) replication system using Escherichia coli cells immobilized in a matrix of agarose have been described (33,34). DNA synthesis in this in vitro system closely resembles DNA replication in vivo in an adenosine 5'-triphosphate (ATP)-dependent process, as shown by the criteria of semiconservative synthesis, reduced synthesis in E. coli dna mutants (mutants thermosensitive for DNA replication [10]), and inhibition of in vitro DNA synthesis by specific inhibitors of DNA replication. In a similar DNA synthesis system using cells immobilized on a planar surface of cellophane, Schaller et al. (28) have shown that one or more nondialyzable components must be present at in vivo concentrations for replicative in vitro DNA synthesis to proceed. The agarose matrix 'Present address: Department of Biochemistry, University of Massachusetts, Amherst, Mass. 01002. thus appears to serve two purposes: (i) it maintains the intactness of the fragile DNA replication complex of DNA, intracellular site, and other components (31, 33); and (ii) it retains certain macromolecular components essential for replicative in vitro DNA synthesis at the required high c...

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Section: Resultsmentioning

confidence: 99%

Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli

Smith¹,

Boerner²

1975

J Bacteriol

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“…The alternatives of chloramphenicol acting as an antagonist of the peptidyl acceptor or of the peptidyl donor have been tested conclusively through an analysis of the reaction products o~E. coli ribosome-poly A system which synthesizes oligolysine (Julian, 1965). --chloramphenicol does not interfere with the formation of lysyl-lysine from two molecules of lysyl-tRNA and, in fact, causes an accumulation of this dipeptide.…”

Section: A Structural Model Of the Chloramphenicol Receptormentioning

confidence: 99%

“…We suggest that chloramphenicol preferentially attaches itself to the newly abandoned P-site, trapping the nascent peptide on the A-site and preventing translocation. This mechanism explains why the drug leads to the formation of dipeptide in an isolated system which normally synthesizes up to octapeptides (Julian, 1965). …”

Section: Model For Chloramphenicol Inhibition Of Transpeptidationmentioning

confidence: 99%

A Structural Model of the Chloramphenicol Receptor Site

Hahn¹,

Gund²

1975

Drug Receptor Interactions in Antimicrobial Chemotherapy

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I . I NTRODUCTI ONHaving a relatively simple chemical structure, chloramphenicol became the first antibiotic to be synthesized by organic-chemical methods (Controulis, Rebstock and Crooks, 1949). A large number of chloramphenicol derivatives was subsequently prepared. Kolosov, Shemiakin, Khokhlov and Gurevich tabulated, in 1961, more than 500 such compounds, and the total number of known chloramphenicol derivatives is, by now, much greater.For many of these substances, data on their growth-inhibitory potencies for bacteria have been reported. Also, after it was recognized that chloramphenicol acts through specific inhibition of bacterial protein biosynthesis (Hahn and Wisseman, 1951; Gale and Folkes, 1953; Wisseman, Smadel, Hahn and Hopps, 1954) and cell-free model systems of protein synthesis were subsequently developed, chloramphenicol derivatives have been tested for inhibitions of such systems. Hence, the chloramphenicol series of compounds appears to be suited to the derivation of structure-activity relationships.The recent identification of a chloramphenicol binding protein which is a constituent of the 50s subunit of 70s ribosomes (Nierhaus and Nierhaus, 1973;Pongs, Bald and Erdmann, 1973) has renewed our interest in these structureactivity relationships and has encouraged us to analyze them with a view to deriving a structural model of a chloramphenicol receptor site which we infer to be located on the peptidyl transferase which is part of the 50s ribosomal subunit. II. STRUCTURE-ACTIVITY RELATIONSHIPS WHICH DETERMINE INHIBITION OF BACTERIAL GROWTHFirst systematic efforts to derive for chloramphenicol derivatives structureactivity rules for bacterial growth inhibition were made by Hahn (1955), Shemiakin, Kolosov, Levitov, Germanova, Karapetian, Shvetsov and Bamdas (1955, 1956) and Hahn, Hayes, Wisseman, Hopps and Smadel (1956). Both groups of workers arrived independently at the same conclusions; reviews of these conclusions represented the state of the art for several years (Kolosov et al., 1961;Hahn, 1967a). Both groups also called attention to the fact that data-on growth inhibitions by chloramphenicol derivatives had been obtained in many laboratories using varieties of methods and test organisms and could not be considered to be better than semi-quantitative approximations which did not lend themselves to the derivation of quantitative structure-activity relationships.

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“…Such high concentrations are necessary to inhibit polyuridylic acid (polyU)-or polyadenylic acid (polyA)-directed in vitro amino acid incorporation (7,9). However, much lower concentrations (10-5 M) of chloramphenicol are sufficient to give equivalent inhibition of bacterial cell growth or in vitro synthesis of peptides stimuLlated by polyC (6), some polynucleotide copolymers (8), or natural messenger (12).…”

mentioning

confidence: 99%

“…The high-speed supernatant fraction (S-100) was used to prepare a pH5 enzyme fraction by the method of Julian (7). Ribosome wash factors were extracted from E. coli MRE 600 ribosomes with 1.0 M NH4Cl in 20 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.8, 2 mm magnesium acetate, and 2 mM dithiothreitol.…”

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confidence: 99%

Effect of Ribosomal Wash Factors on Inhibition by Chloramphenicol

Cameron

Rogers

Julian

1972

Antimicrob Agents Chemother

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In vitro protein-synthesizing systems from Escherichia coli can be categorized as either chloramphenicol-sensitive or chloramphenicol-insensitive. The chloramphenicol-sensitive systems used in this study required the presence of factors removed from ribosomes with 1.0 M NH4Cl when chromatographically purified ribosomes were used for amino acid incorporation. These ribosomal wash factors inhibited but did not eliminate amino acid incorporation in chloramphenicol-insensitive systems. For both systems, addition of increasing amounts of the ribosomal wash factors increased the sensitivity to chloramphenicol inhibition.Previous studies on the mechanism of action of chloramphenicol have often utilized high concentrations (10-4 to 10-3 M) of the drug, and have shown inhibition of chain elongation. Such high concentrations are necessary to inhibit polyuridylic acid (polyU)-or polyadenylic acid (polyA)-directed in vitro amino acid incorporation (7, 9). However, much lower concentrations (10-5 M) of chloramphenicol are sufficient to give equivalent inhibition of bacterial cell growth or in vitro synthesis of peptides stimuLlated by polyC (6), some polynucleotide copolymers (8), or natural messenger (12).Elongation, in contrast to initiation or release, is thought to involve the same steps regardless of the messenger used. Such differential sensitivity is, therefore, difficult to account for if low con-.centrations of chloramphenicol affect sensitive systems in the same way as high levels affect insensitive systems.Protein-synthesizing systems using purified ribosomes normally utilized for polyA-and polyU-stimulated incorporation systems are deficient in ribosomal wash factors required for natural messenger translation. This report deals with the stimulating effect of these factors on polyC-directed incorporation of ['4C]proline, and with the effect of these factors on enhancement of inhibition by chloramphenicol of polyA-and polyU-stimulated incorporation.

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[14C]Lysine peptides synthesized in an in vitro Escherichia coli system in the presence of chloramphenicol

Cited by 55 publications

References 11 publications

Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli

Stimulation of adenosine 5'-triphosphate-dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli

A Structural Model of the Chloramphenicol Receptor Site

Effect of Ribosomal Wash Factors on Inhibition by Chloramphenicol

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