Stimulation of 14C-leucine incorporation into protein In vitro by ribosomal RNA of Escherichia coli

Otaka, Eiko; Osawa, Syozo; Sibatani, Atuhiro

doi:10.1016/0006-291x(64)90506-6

Cited by 49 publications

(9 citation statements)

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“…The DNA from this transducing phage has been shown to be a good template for the synthesis of rRNA (R. Almendinger and M. Nomura, unpublished experiments). Thus absence of r-protein synthesis with this DNA as a template appears to exclude the hypothesis (19,20) that "nascent" rRNAs are the templates for r-proteins. However, it is still possible that nascent rRNAs have some role in the synthesis of r-proteins.…”

Section: Resultsmentioning

confidence: 92%

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

Kaltschmidt

Kahan

Nomura

1974

Proc. Natl. Acad. Sci. U.S.A.

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In vitro synthesis of a number of E. coli 30S ribosomal proteins has been demonstrated in a cell-free system consisting of ribosomes, initiation factors, RNA polymerase, a fraction containing soluble enzymes and factors, and E. coli DNA. DNA-dependent synthesis of the following 30S proteins has been demonstrated: S4, S5, S7, S8, S9, SlO, S13, S14, S16, S19, and S20. (4), and Schweiger and Gold (5). In preliminary experiments we obtained better results using the former system (4, 6); we therefore decided to modify that system. Our major modification was to fractionate the "S30" of the Zubay system into salt-washed ribosomes, an initiation factor fraction ("IF"), a partially purified "enzyme fraction" ("SlOODE"), and DNA.-dependent RNA polymerase. S1OODE contains EF-T, EF-G, and aminoacyl-tRNA synthetases, and is devoid of nucleic acids. Amino-acid incorporation in the absence of added DNA is very small (see [35S ]Methionine was incorporated into protein with E. coli DNA as a template, essentially as described in Table 1. After 2 hr of incubation, the reaction mixtures were cooled and dialyzed against TRI buffer containing 8 M urea and then TMAI buffer containing 8 M urea. This treatment dissociated the ribosomes in the incubation mixture so that the released r-proteins could mix with radioactive r-proteins synthesized in the system. The resultant solution was passed through a DEAE-cellulose column (1 X 10 cm, equilibrated with 8 M urea in TMAI buffer, pH 7.4). Most of the 30S r-proteins lassed through the column. However, the "acidic" r-proteins, S1, S2, and S6, were probably retained together with other "soluble proteins" and nucleic acids (9). The pass-through fraction ("basic protein fraction") contained about 50% of the total proteins.Carrier nonradioactive 30S r-protein mixture (TP30) and 16S RNA were then added to the basic protein fraction, and 30S ribosome reconstitution was performed (7,8

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Section: Resultsmentioning

confidence: 92%

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

Kaltschmidt

Kahan

Nomura

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…Since the stability of rRNA apparently depends on the degree of methylation, undermethylated rRNA is expected to be unstable and undergo turnover. Moreover, since mRNA, unlike rRNA, is not methylated, the hypothesis can be formulated that RNA function also depends on the degree of methylation (see Otaka, Osawa & Sibatani, 1964) and will be altered by virginiamycin.…”

Section: Cocito Discussionmentioning

confidence: 99%

Metabolism of Macromolecules in Bacteria Treated with Virginiamycin

Cocito¹

1969

Journal of General Microbiology

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SUMMARYThe two components of virginiamycin, M and S, separately exerted a reversi ble bacteriostatic activity on Bacillus subtilis. Their combination increased by a hundredfold the inhibitory activity of each factor and induced a loss of viability of bacteria. Such an irreversible step was preceded by a reversible phase, which was characterized by a long lag in colony formation.Very short incubation with single virginiamycin components and their combination suddenly and completely blocked protein synthesis, whereas the rate of incorporation of labelled bases and nucleosides into polynucleotides was not altered appreciably unless protein formation was halted completely.Nevertheless, some alterations of ribosomal RNA metabolism occurred very early after treatment with virginiamycin. The synthesis of 23s rRNA was specifically inhibited. Moreover, the degree of methylation of the rRNA which was made in the presence of the drug was lower than that of the controls. Also, the rRNA labelled in virginiamycin-treated cells was metabolically unstable. This indicates that formation and stability of rRNA, as well as the balance among rRNA species, depend on virginiamycinsensitive protein synthesis.Metabolism of pulse-labelled RNA was also altered in the presence of virginiamycin : its half-life was prolonged about sixfold by single components and eightfold by their combination. This was due to an increased turnover of rRNA and to prevention of messenger RNA decay.It is concluded that peptide chain formation is the primary target of Virginiamycins M and S (hence their synergistic antibiotic activity), that translationnot transcription-is prevented by these inhibitors, and that the alterations of nucleic acid metabolism are due to the halt of protein synthesis.

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“…(1965) in RNA preparations obtained by fractionation based on temperature increases during extraction, using Ehrlich ascites cells. Our finding that template activity was associated with these fractions might be accounted for by a hypothesis on the transient template functions of ribosomal RNA precursors (Roberts, 1965 ;Otaka et a/., 1964).…”

Section: Figurementioning

confidence: 79%

“…These components, found for the first time in a study of rapidly labelled RNA from HeLa cell nuclei (Scherrer et a/ (Roberts, 1965 ;Otaka et a/., 1964).…”

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confidence: 93%

RNA with high template activity from leukaemic myeloblasts (Bai strain a virus‐induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid ³²P labelling

Veprek

Trávnícek

Ríman

1967

Intl Journal of Cancer

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R N A preparations made from leukaemic myeloblasts by fractionating extraction in a twoFurther elucidation of the mechanisms through which the template-active (Riman et al., 1967a, b) RNA of the leukaemic virus (BAI strain A virus, avian myeloblastosis; Eckert et al., 1951) acts at the level of active protein-synthesizing structures, would require means for the isolation, detection and separation of the template-active RNAs of the cell and of the virus. Such techniques would also be necessary for distinguishing between these and other metabolically active cellular RNAs, as well as for the study of the structural localization of functionally active virus RNA in the infected cell. Some of the above prerequisites for the study of the functional role of viral RNA in subcellular systems were met in our previous paper (TravniEek et al., 1967) in which a suitable method was selected and the optimum conditions for isolating highly template-active cellular RNAs were determined. The paper presented here extends further this information by defining the sedimentation profile of the template-active

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Stimulation of 14C-leucine incorporation into protein In vitro by ribosomal RNA of Escherichia coli

Cited by 49 publications

References 3 publications

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

Metabolism of Macromolecules in Bacteria Treated with Virginiamycin

RNA with high template activity from leukaemic myeloblasts (Bai strain a virus‐induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid ³²P labelling

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Stimulation of 14C-leucine incorporation into protein In vitro by ribosomal RNA of Escherichia coli

Cited by 49 publications

References 3 publications

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

In Vitro Synthesis of Ribosomal Proteins Directed by Escherichia coli DNA

Metabolism of Macromolecules in Bacteria Treated with Virginiamycin

RNA with high template activity from leukaemic myeloblasts (Bai strain a virus‐induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid 32P labelling

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RNA with high template activity from leukaemic myeloblasts (Bai strain a virus‐induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid ³²P labelling