Two Classes of Membrane Binding of Replicative RNA of Bacteriophage MS2

Abstract

Escherichia coli membranes were isolated in the presence of 6 mM Mg++. (1, 2). These syntheses are organized in the cell to produce yields of infectious virus as high as 104 per cell, but enzyme preparations give net synthesis of infectious RNA of only 10-fold (3) or less (4, 5). The difference in the in vivo and in vitro yield of phage RNA could be explained if the synthesis were tightly ordered in the cell. Use of membrane as a site of RNA synthesis should increase the efficiency of synthesis by providing a surface for adsorption of precursors, spatial organization of sequential steps, and limitation of the possible configurations of the RNA to those that are the most favorable for synthesis and assembly. The observation that a histidine-containing MS2-specific protein is associated with a rapidly sedimenting cell fraction orginally led to the suggestion that MS2 RNA synthesis could be organized on membrane (6). Further work (7) showed that labeled uracil is first incorporated in the presence of actinomycin D into MS2-specific RNA associated with a fraction sedimenting at 30,000 X g in the presence of 6 mM Mg++. This RNA was incorporated into a virion precursor particle (7) or organized into polyribosomes (unpublished data) before its release into the "supernatant." The rapidly sedimenting fraction was thought to be membrane because it was solubilized by detergents and urea (7,8) and because it contained cell lipid (8).Electron microscopic studies confirmed that this fraction contained membrane (8). This membrane preparation differs from many in that no detergents were used and 6 mM Mg++ was continuously present. When the rapidly sedimenting cell fraction is washed with buffer lacking Mg++ (or other divalent cations) some components are released, "membrane eluate," while others still sediment, "membrane." This work describes the kinetics of labeling of the different MS2 RNA species in the three cell fractions, supernatant, membrane eluate, and membrane. MATERIALS AND METHODSBacteria and Growth Medium have been described (7). Bufers. TKA buffer is 10 mM Tris HCl (pH 8)-40 mM KCI-10 mM NaN3. TMKA buffer TKA plus 6 mM MgC12.Cell Fractions. To harvest cells, NaN3 (10 mM) and unlabeled uracil (100 ,ug/ml) were added to 200-ml aliquots of cells at a concentration of 2 X 108/ml. These were rapidly chilled by swirling in a wide-bottom flask in a dry-ice-alcohol bath. The following steps were done at 0-4'. The cells were centrifuged and suspended in 0.6 ml of TMKA buffer. The success of the lysis depends upon a high cell concentration.Lysozyme (1 mg/ml) was added, and after 3 min, the cells were rapidly frozen and thawed twice. Electrophoretically purified DNase (10 /ug/ml) was added, and at least 10 min later, the lysate was centrifuged at 30,000 X g for 20 min. The resulting sediment was resuspended twice in TMKA buffer with vigorous mixing on a Vortex mixer and centrifuged at 30,000 X g for 20 min. The resultant supernatants were combined and referred to as "supernatant." The resultant sediment was resuspend...

Effect of an Escherichia coli traD (ts) Mutation on MS2 RNA Replication

“…These changes might be responsible for the inhibition of MS2 RNA replication in the mutant at 42 °C. This idea is supported by previous studies showing that MS2 replicative intermediates are tightly bound to the membrane fraction (Haywood, 1973). We suggest that group I RNA replication, in contrast to that of Qfl, takes place on a membrane structure in which the traD gene product is either directly or indirectly involved.…”