Interaction of Escherichia coli 30S Ribosomal Subunits with MS2 Phage RNA in the Absence of Initiation Factors

Abstract

(ATCC 19089) were laboratory strains; Bacillus brevis Vm4 was kindly provided by Z. Kurylo-Borowska, the Rockefeller University. Cells were grown as described (13,15,16) and harvested at mid-logarithmic growth phase.Ribosomes and Ribosomal Subunits. These were obtained by procedures established for E. coli, and are described in recent publications (12, 13). Unfractionated crude ribosomes were washed twice (15 hr and 4-5 hr) in a buffer [20 mM Trisacetate (pH 7.8), 10 mM magnesium acetate, 0.1 mM dithiothreitol] containing 1.0 M NH4C1. To ascertain whether the 30S ribosomal subunits were fully devoid of initiation 'factors, each batch of washed 70S ribosomes was assayed before fractionation into subunits (Table 1). It was particularly important to use IF-3-free ribosomes. As seen in Table 1,

Lack of ribosomal protein S1 in Bacillus stearothermophilus.

“…They did not crossreact with the antiserum against E. coli S1 at all the concentrations (0. [5][6][7][8][9][10] ,ug per well) tested. Similarly, two other acidic proteins of B.…”

“…The difference between S-30S and F-SOS was thus concluded to be only the presence in the former and absence from the latter of the protein Si. MS2 phage RNA was found (7) to bind only to the former..…”

“…Protein Si is the largest of all the ribosomal proteins of E. coli (2) and has been considered to be a fractional protein, that is, a protein present less than one copy per ribosome (3,4). Recently, this protein was shown to be indispensable for the translation of natural as well as synthetic messengers (5,6) and for the binding of MS2 phage RNA to ribosomes (7). The 30S ribosomal subunit of E. coli was shown (7)(8)(9) to exist in at least two forms, a fast migrating form (F-3OS) and a slow migrating form (S-30S), which were separable from each other by electrophoresis on agaroseacrylamide composite gels.…”

“…Electrophoresis on 0.5% agarose-3% acrylamide composite gels in Tris-boric acid-EDTA buffer, pH 8.3, was performed as described (7,8). Gels were either stained with 0.2% methylene blue or cut into i-mm slices, swelled in Soluene 350 (Packard), and their radioactivity determined.…”

Interaction of Neomycin with Ribosomes and Ribosomal Ribonucleic Acid

“…Electrophoresis of ribosomes in an EDTA-containing gel causes dissociation of the subunits and release of 5S RNA (5 (4,17) were unaffected by addition of neomycin (Fig 1, 3, and 6). Reversibility of neomycin binding to RNA.…”