Supplementation of the growth medium with high concentrations of NaCl, KCl, or sucrose caused a drastic change in the ratio of the two peptidoglycanassociated major outer membrane proteins of Escherichia coli K-12 in that the amounts of proteins b and c present in cell envelope preparations decreased and increased, respectively. Kinetic studies showed that, after the osmolarity of the medium was changed, one protein was hardly incorporated into the membrane, whereas the other was incorporated with an increased rate. After about 1.5 to 2 generations, the cell envelopes obtained the b/c ratio characteristic for the new medium, and both proteins were subsequently incorporated with rates that ensured this new ratio. Once proteins b and c were incorporated in the cell envelope, they were not converted into each other by changes in osmolarity of the growth medium
1. The binding of washed 30-S ribosomal subunits of Escherichia coli to MS2 RNA in the absence of fMet-tRNA requires the presence of the initiation factor IF-3. No binding is observed with IF-1, IF-2 or a combination of these two initiation factors.2. The IF-3-dependent binding is stimulated about two-fold by IF-2. IF-1 has no effect in this respect. Optimal binding occurs a t about 7 mM Mg2+.3. Upon incubation of [35S]IF-3, MS2 [3H]RNA and 30-S subunits, complexes are formed which contain the three components in a 1 : 1 : 1 ratio. These ternary complexes have a sedimentation coefficient of about 40 S and a buoyant density in CsCl of about 1.74 g / d .4. The ternary complexes formed in the absence of IF-2 and IF-1 are rather labile and readily dissociate into MS2 RNA and IF-3-containing ribosomes. Their half-life a t 0 "C is about 40 min. I n the presence of IF-2 and IF-1, complexes are formed which remain stable for a t least 6 h. Unwashed native 30-S subunits also form stable complexes with MS2 RNA.5. Binding of washed 30-S subunits to unfolded MS2 RNA (MS2 RNA treated with formaldehyde) does not require initiation factors. Complexes containing more than one ribosomal particle per messenger can be formed. Attachment of fMet-tRNA to these complexes requires IF-2 and IF-1, but is optimal in the presence of all three initiation factors. . Initiation factors therefore promote mRNA binding to 30-5 ribosomal subunits independently from their effect on fMet-tRNA binding to this particle. This has been shown also for the attachment of 30-S E. coli ribosomal subunits to the plant viral messenger derived from alfalfa mosaic virus [3].Abbreviation. Brij 58, polyoxyethylene cetylether. Definition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 260 nm, when measured in a 1-cm path-length cell.Using an electron microscopic method for quantitative measurements of ribosome binding to nascent T4 rnRNA, Revel et at. showed that IF-2 alone stimulates 3 0 3 subunit attachment to T4 mRNA. The two other factors had little effect by themselves [4]. Similar experiments with mRNA from bacteriophage f2, MS2 or Qj3 were hampered by the fact that this RNA by itself sediments at 2 8 s and that complexes with 30-S subunits could not be demonstrated unambiguously. The formation of a small amount of such a putative complex in the presence of IF-1 and IF-3 has been reported by Sabol et al. [5], but the evidence was only suggestive.That IF-3 is specifically required for the recognition of initiation sites on the messenger has been shown in a number of laboratories [6-151. I n particular the finding that distinct IF-3 factors can be isolated, one recognizing E. coli mRNA, MS2 RNA and early T4 mRNA, the other recognizing late T4 mRNA, clearly illustrates the ability of IF-3 to direct the 3 0 3 ribosomal subunit to specific messenger sites. Eur. J. Biochem. 40 (1973)
SummaryThe hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and aqueous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.
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