Abstract
A two-step procedure has been described for the total reconstitution of 50S ribosomal subunit from E. coli. RNA and proteins are mixed with stoichiometry of 1:1.2 and incubated at 44 degrees C in 4.0 mM Mg2+ followed by a second incubation at 50 degrees C in 20 mM Mg2+ (Dohme and Nierhaus, J. Mol. Biol. 107, 585 (1976)). A modified method recently reported makes use of an altered preparation technique for the RNA and proteins and requires an RNA to protein stoichiometry of 1:2.5 and 7.5 mM Mg2+ in the first incubation (Amils et al., Nucl. Acid Res. 5, 2455 (1978)). The latter requirements are not compatible with the findings obtained with the first procedure. A comparison of the various RNA and protein fractions from the different groups revealed that the Mg2+ dependence of reconstitution is a function of the RNA preparation, whereas the stoichiometry depends upon the technique used for isolation of the protein fraction. The different RNA preparations were compared in the electron microscope.
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- Amils R., Matthews E. A., Cantor C. R. An efficient in vitro total reconstitution of the Escherichia coli 50S ribosomal subunit. Nucleic Acids Res. 1978 Jul;5(7):2455–2470. doi: 10.1093/nar/5.7.2455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dohme F., Nierhaus K. H. Role of 5S RNA in assembly and function of the 50S subunit from Escherichia coli. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2221–2225. doi: 10.1073/pnas.73.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dohme F., Nierhaus K. H. Total reconstitution and assembly of 50 S subunits from Escherichia coli Ribosomes in vitro. J Mol Biol. 1976 Nov 15;107(4):585–599. doi: 10.1016/s0022-2836(76)80085-x. [DOI] [PubMed] [Google Scholar]
- Nierhaus K. H., Dohme F. Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4713–4717. doi: 10.1073/pnas.71.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nierhaus K. H., Montejo V. A protein involved in the peptidyltransferase activity of Escherichia coli ribosomes. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1931–1935. doi: 10.1073/pnas.70.7.1931. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schreiner G., Nierhaus K. H. Protein involved in the binding of dihydrostreptomycin to ribosomes of Escherichia coli. J Mol Biol. 1973 Nov 25;81(1):71–82. doi: 10.1016/0022-2836(73)90248-9. [DOI] [PubMed] [Google Scholar]
- Sieber G., Nierhaus K. H. Kinetic and thermodynamic parameters of the assembly in vitro of the large subunit from Escherichia coli ribosomes. Biochemistry. 1978 Aug 22;17(17):3505–3511. doi: 10.1021/bi00610a013. [DOI] [PubMed] [Google Scholar]
- Spillmann S., Dohme F., Nierhaus K. H. Assembly in vitro of the 50 S subunit from Escherichia coli ribosomes: proteins essential for the first heat-dependent conformational change. J Mol Biol. 1977 Sep 25;115(3):513–523. doi: 10.1016/0022-2836(77)90168-1. [DOI] [PubMed] [Google Scholar]
- Spillmann S., Nierhaus K. H. The ribosomal protein L24 of Escherichia coli is an assembly protein. J Biol Chem. 1978 Oct 10;253(19):7047–7050. [PubMed] [Google Scholar]
- Teraoka H., Nierhaus K. H. Proteins fro Escherichia coli ribosomes involved in the binding of erythromycin. J Mol Biol. 1978 Dec 5;126(2):185–193. doi: 10.1016/0022-2836(78)90358-3. [DOI] [PubMed] [Google Scholar]
- Yamada T., Mizugichi Y., Nierhaus K. H., Wittmann H. G. Resistance to viomycin conferred by RNA of either ribosomal subunit. Nature. 1978 Oct 5;275(5679):460–461. doi: 10.1038/275460a0. [DOI] [PubMed] [Google Scholar]