Abstract
Cell-free Staphylococcus aureus extracts have been prepared which actively incorporate amino acids into protein. The requirements for amino acid incorporation of this preparation were strongly suggestive of de novo protein synthesis, since it showed an absolute requirement for ribosomes, 105,000 × g supernatant fluid, energy source, and magnesium ion. The stability of these extracts was greatly improved by use of dithiothreitol instead of mercaptoethanol as a sulfhydryl protecting reagent. Data were presented to show that the binding of aminoacyl-soluble ribonucleic acid to ribosomes did not require guanosine triphosphate and supernatant enzyme. The major characteristic which distinguishes this system from other cell-free systems is the much higher magnesium concentration required to maintain ribosomes intact and to obtain the maximal incorporation of amino acids. Addition of polyuridylic acid, polyadenylic acid, or polycytidylic acid caused about 60-fold, 30-fold, or 4-fold stimulation of the incorporation of phenylalanine, lysine, or proline, respectively. Studies by density gradient sedimentation indicated that radioactive polyuridylic acid or polyadenylic acid was associated with the monosomes. This complex can actively synthesize polypeptides. On the other hand, the nascent protein synthesized under the direction of endogenous messenger ribonucleic acid was associated with both polysomes and monosomes.
Full text
PDF
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- BOARDMAN N. K., FRANCKI R. I., WILDMAN S. G. PROTEIN SYNTHESIS BY CELL-FREE EXTRACTS FROM TOBACCO LEAVES. II. ASSOCIATION OF ACTIVITY WITH CHLOROPLAST RIBOSOMES. Biochemistry. 1965 May;4:872–876. doi: 10.1021/bi00881a012. [DOI] [PubMed] [Google Scholar]
- Barondes S. H., Nirenberg M. W. Fate of a Synthetic Polynucleotide Directing Cell-Free Protein Synthesis I. Characteristics of Degradation. Science. 1962 Nov 16;138(3542):810–813. doi: 10.1126/science.138.3542.810. [DOI] [PubMed] [Google Scholar]
- GALE E. F., FOLKES J. P. The assimilation of amino acids by bacteria. 20. The incorporation of labelled amino acids by disrupted staphylococcal cells. Biochem J. 1955 Apr;59(4):661–675. doi: 10.1042/bj0590661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- GALE E. F., SHEPHERD C. J., FOLKES J. P. Incorporation of amino-acids by disrupted staphylococcal cells. Nature. 1958 Aug 30;182(4635):592–595. doi: 10.1038/182592a0. [DOI] [PubMed] [Google Scholar]
- GARDNER R. S., WAHBA A. J., BASILIO C., MILLER R. S., LENGYEL P., SPEYER J. F. Synthetic polynucleotides and the amino acid code. VII. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2087–2094. doi: 10.1073/pnas.48.12.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- GIERER A. Function of aggregated reticulocyte ribosomes in protein synthesis. J Mol Biol. 1963 Feb;6:148–157. doi: 10.1016/s0022-2836(63)80131-x. [DOI] [PubMed] [Google Scholar]
- GILBERT W. Polypeptide synthesis in Escherichia coli. I. Ribosomes and the active complex. J Mol Biol. 1963 May;6:374–388. doi: 10.1016/s0022-2836(63)80050-9. [DOI] [PubMed] [Google Scholar]
- NATHANS D., LIPMANN F. Amino acid transfer from aminoacyl-ribonucleic acids to protein on ribosomes of Escherichia coli. Proc Natl Acad Sci U S A. 1961 Apr 15;47:497–504. doi: 10.1073/pnas.47.4.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
- NIRENBERG M. W., MATTHAEI J. H. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A. 1961 Oct 15;47:1588–1602. doi: 10.1073/pnas.47.10.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
- NIRENBERG M., LEDER P. RNA CODEWORDS AND PROTEIN SYNTHESIS. THE EFFECT OF TRINUCLEOTIDES UPON THE BINDING OF SRNA TO RIBOSOMES. Science. 1964 Sep 25;145(3639):1399–1407. doi: 10.1126/science.145.3639.1399. [DOI] [PubMed] [Google Scholar]
- SZER W., OCHOA S. COMPLEXING ABILITY AND CODING PROPERTIES OF SYNTHETIC POLYNUCLEOTIDES. J Mol Biol. 1964 Jun;8:823–834. doi: 10.1016/s0022-2836(64)80163-7. [DOI] [PubMed] [Google Scholar]
- TAKANAMI M., OKAMOTO T. INTERACTION OF RIBOSOMES AND SYNTHETIC POLYRIBONUCLEOTIDES. J Mol Biol. 1963 Oct;7:323–333. doi: 10.1016/s0022-2836(63)80027-3. [DOI] [PubMed] [Google Scholar]
- Tissieres A., Schlessinger D., Gros F. AMINO ACID INCORPORATION INTO PROTEINS BY ESCHERICHIA COLI RIBOSOMES. Proc Natl Acad Sci U S A. 1960 Nov;46(11):1450–1463. doi: 10.1073/pnas.46.11.1450. [DOI] [PMC free article] [PubMed] [Google Scholar]