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)
A kinetic study of induction of the enzymes of the lactose operon was carried out under conditions known to affect the kinetics of derepression of the enzymes of the histidine operon. The results show that the lactose system is similar to the histidine system in its responsiveness to conditions thought to affect the formylating capacity of the cell. This was demonstrated in the following ways: (i) trimethoprim, which is known to reduce the formylating capacity of the cell, gives rise to a relatively long interval between the times of induction of 3-galactosidase and transacetylase; (ii) under conditions in which the histidine operon is derepressed, chloramphenicol causes a prolongation of the interval between the times of induction of the two enzymes, and this prolongation is reversed by adenine, methionine, and serine, compounds known to enrich the one-carbon pool of the cell; and (iii) 4-amino-5-imidazolcarboxamide ribonucleoside, a compound which may act as a drain for formyl groups, reverses the effect of the latter compounds. The finding that the interval between the times of induction of the two enzymes is shortened under conditions expected to maintain a relatively high intracellular fo rmylating capacity suggests that under certain conditions translation of the polycistronic messenger ribonucleic acid of the lactose operon may be initiated at more than one site or may proceed more rapidly from the operator end. From previous studies on the kinetics of derepression of the histidine enzymes in a wide variety of histidine auxotrophs of Salmonella typhimurium, it was concluded that derepression may occur either simultaneously or sequentially, depending upon the formylating capacity of the cell (8). The enzymes became derepressed simultaneously in those mutants with an adequate formylating capacity, whereas they became derepressed in a temporal sequence, corresponding to the order of the genes in the histidine operon, in those mutants with a reduced formylating capacity. The reason for the differences in formylating capacity among the various histidine auxotrophs is that those mutants with a block in any of the last four steps of the histidine pathway produce phosphoribosyl aminoimidazole carboxamide (PR-AIC) as a byproduct of the path
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