Preparation procedures of proteins and RNA influence the total reconstitution of 50S subunits from E. coli ribosomes.
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.
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008274:Magnesium; D008722:Methods; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D008274:Magnesium