Inhibition of ribosomal peptidyltransferase by chloramphenicol. Kinetic studies

Drainas, Denis; Kalpaxis, Dimitrios L.; Coutsogeorgopoulos, C.

doi:10.1111/j.1432-1033.1987.tb10991.x

Cited by 59 publications

(37 citation statements)

References 33 publications

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“…The mechanism by which chloramphenicol and p-aminosalicylate inhibit the Mg-chelatase activity is not clear. Chloramphenicol binds to ribosomes and interferes with the peptidyl transferase activity (Drainas et al 1987). p-Aminosalicylate in millimolar concentrations is known to disrupt protein RNA interactions (Parish and Kirby 1966).…”

Section: Discussionmentioning

confidence: 99%

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Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

et al. 1997

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Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272,000 x g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.

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Section: Discussionmentioning

confidence: 99%

“…2C). Chloramphenicol in micromolar concentrations is known to inhibit protein synthesis (Drainas et al 1987).…”

Section: Barley Mg-chelatasementioning

confidence: 99%

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

et al. 1997

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“…The action of the aminoglycosides is presumably targeted against more than one single cellular process [9]. From the literature, Chl prevents the peptidyltransferase reaction [10]; Ery is believed to act by blocking the translocation of peptidyl-tRNA from the A to the P site [11]; Fus inhibits the same step by binding to Elongation factor G [12], and Tet prevents binding of aminoacyl-tRNA mainly to the A site [13]. Finally, Pur is an aminoacyl-adenosine analog and leads to premature chain termination [11].…”

Section: Introductionmentioning

confidence: 99%

Mechanism‐related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional and translational inhibitors

Evers¹,

Padova²,

Meyer³

et al. 2001

Proteomics

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High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemophilus influenzae extracts allows for the separation and quantification of more than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transcription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromycin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin (Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggered a clear increase in the synthesis rates of ribosomal proteins and RNA polymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antitermination as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chl, Ery, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chl, Ery, Fus, Rif, and Tet resulted in a decrease in the concentration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.

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“…Crystallographic studies showed that chloramphenicol binds either to the hydrophobic crevice to the entrance of the peptide exit tunnel of Haloarcula marismortui [39], or to the active site hydrophobic crevice of Deinococcus radiodurans ribosomal subunits [40], suggesting that both of these sites may be physiologically relevant. It was suggested that these two different chloramphenicol binding sites observed in the structural studies of the archaeal (H. marismortui) and eubacterial (D. radiodurans) 50S subunits most likely correspond to the two binding sites on eubacterial 50S subunits that have been inferred previously from binding [41] and kinetic studies [42,43].…”

Section: Effect Of Small Rna Ligands On Ribosomementioning

confidence: 94%