Membrane protein synthesis after infection of Escherichia coli B with phage T4: The rIIB protein

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 This RNA was incorporated into a virion precursor particle (7) or organized into polyribosomes (unpublished data) before its release into the "supernatant."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...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Two Classes of Membrane Binding of Replicative RNA of Bacteriophage MS2

Haywood¹

1973

“…The r mutants do not elicit the phenomenon of lysis inhibition (11) and are incapable of growing in E. coli cells lysogenic for phage X (15). It has already been shown (16,17) …”

mentioning

confidence: 99%

Association of the r IIA Protein with the Bacterial Membrane

Ennis

Kievitt

1973

Self Cite

The Escherichia coli cell envelope is modified after infection with wild-type phage T4. For example, the cells become resistant to lysis from without (1), can be lysis inhibited (2), exclude superinfecting T4 phage (3) and ghosts (4), become increasingly permeable to ions and other compounds (5-7), and increase their synthesis and hydrolysis of phospholipids (8)(9)(10). At least some of these functions are coded for by the phage, because specific phage mutants, such as r (11), spackle (12), ac (7), and imm (13) affecting these properties of the membrane, have been isolated. Furthermore, some of these changes require protein synthesis for their elucidation, indicating that phage-coded proteins are involved in membrane modification (13,14).One of these membrane changes is coded for by the rnI locus, which is composed of two cistrons, A and B (15). The r mutants do not elicit the phenomenon of lysis inhibition (11) and are incapable of growing in E. coli cells lysogenic for phage X (15). It has already been shown (16,17)

“…It is, however, partially functional in its interaction with the replication complex. ( Since the rII proteins are bound to the membrane (34)(35)(36) and to DNA (37), the rII-suppression of L171 implies that the conversion of "joint" into "recombinant" molecules occurs at the membrane. This implication is strengthened by the suppression of the rII-plaque morphology by some gene 32 (and ligase) mutations.…”

Section: Discussionmentioning

confidence: 99%

Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

Mosig¹,

Breschkin²

1975

Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to (6,(9)(10)(11). It is produced in large amounts after T4 infection and is limiting for phage production (12). This could mean that (i) DNA replication, recombination, and repair are interdependent, and although gene 32 protein is directly involved in only one of these processes, it indirectly affects the other two; (ii) the protein is directly involved in all three processes and serves the same function in each; or (iii) the protein serves a direct but different function in each of the three processes.We have found that different gene 32 ts mutants have quite different phenotypes, and that at least three, L171 being one of them, are partially suppressed by rII mutations (ref.