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Nucleic Acids Research, 1980, Vol. 8, No. 5 989-998
© 1980

Articles

Preparation procedures of proteins and RNA influence the total reconstitution of 5OS subunits from E.coli ribosomes

Volker Nowotny, Hans-Jörg Rheinberger, Knud Nierhaus*, Bernd Tesche{dagger} and Ricardo Amils+

Max-Planck-lnst. Molekulare Genetik Abt. Wittmann, Ihnestr. 63–73, Berlin-Dahlem {dagger}Teilinst. Elektronenmikroskopie, Fritz-Haber-Inst., Max-Planck-Gesellschaft Faradayweg 4–6, Berlin-Dahlem, GFR +Cent. Biologia Molécular, Universidad Autónoma de Madrid Madrid-34, Spain

*to whom correspondence should be addressed

Received December 21, 1979. 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°C in 4.0 mM Mg2+ followed by a second incubation at 50°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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