Febs Letters Volume 49, Number 1-3./Volume 50, Number 1-3./Master Index Volumes 41-50. [antikvár]

Volume 49, number 1 FEBS LETTERS December 1974 THE RELEASE OF PROTEINS AND 5S RNA DURING THE UNFOLDING OF ESCHERICHIA COLI RIBOSOMES R. A. GARRETT, C. SCHULTE and G. STÔFFLER Max Planck Institute ßr Molekulare Genetik, 1 Berlin 33, G.F.R. P. GRAY* Centre de Biochimie et de Biologie Moléculaire, CNRS, Marseille, France and R. MONIER Institut de Recherches Scientifiques sur le Cancer, CNRS, B.P. No. 8, 94800-Villeiuif, France Received 7 August 1974 Revised version received 10 September 1974 1. Introduction When ribosomal subunits from Escherichia coli are dialysed extensively against either low salt concentrations (1 mM EDTA, 5 mM Tris-HCl, pH 7.6), or against high salt (1 M NHiO, 0.1 mM MgQz, 10 mM Tris—HQ, pH 7.6), a more open ribosomal structure is formed that sediments more slowly than the untreated ribosomal subunits [1-6]. This structural change has been termed 'unfolding'. It has been demonstrated that almost all of the 5S RNA is released from the SOS subunits during unfolding m the presence of both EDTA [7] and 1 M NHiQ [3]. Although there is no report, to date, of any proteins bemg co-released during EDTA-unfolding [2], there is evidence of 4—7 unidentified proteins being released by the high-salt treatment [3]. Since it is known that a group of proteins are bound to 5S RNA in the SOS subunit [8-11], and facilitate complex formation of SS RNA with 23S RNA [8,12], we attempted to identify which proteins, if any, are released during unfolding, in order to establish whether they correspond to the proteins that bind to SS RNA. * Present address: Department of Biochemistry and Molecular Biology, University of Oklahoma, Oklahoma 73190, U.S.A. It was found that a few protems were released with the SS RNA during both unfolding procedures, only one of which, L25, binds strongly to SS RNA. The other SS RNA-binding proteins remained attached to the SOS subunit. During high salt unfolding, protein L30 that assembles weakly with SS RNA [10] was also detached. Functional studies, reviewed in [13], suggest that the other proteins that were wholly or partially removed may lie within a functionally important region in the vicmity of SS RNA within the SOS subunit. 2. Materials and methods 2.1. Preparation of unfolded 708 ribosomes and 505 subunits 70S ribosomes and SOS subunits were prepared as described earlier [8]. 2.1.1. EDTA treatment The SOS subunits (or ribosomes) were dissolved in 10 mM Tris-HCl, pH 7.6 and 1 to S mM EDTA at 10-60 mg/ml and treated as follows. 1) The ribosome sample (2-6 ml) was dialysed (16 hr) against this buffer, and passed over an A l.S agarose (Biorad, CaUfornia) column (2 X 180 cm). The ionic strength of the unfolded ribosomes, that were eluted in the excluded volume, was adjusted to 10 mM MgG^ and North-Holland Publishing Company - Amsterdam 1