Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3

Abstract

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)

Interaction of poly(A) and mRNA with eukaryotic initiator met-tRNA-f binding factor: identification of this activity on reticulocyte ribonucleic acid protein particles.

“…Since we have been able to show an interaction of mRNA with the Met-tRNAf binding factor, it is tempting to speculate an association between these events in the initiation process. A relationship between initiator tRNA and messenger RNA binding has been reported previously in bacterial systems (37)(38)(39). Escherichia coli IF-2, the prokaryotic initiation factor which forms a ternary complex with Met-tRNAf (40)(41)(42), also participates with IF-3 in binding mRNA to the 30S ribosomal subunit (37)(38)(39)(40)(41)(42).…”

The Mode of Action of Thiostrepton in the Initiation of Protein Synthesis

“…Furthermore, high amounts of IF-3 resulted in an increase of inhibition, which is hard to reconcile with the observation that when the 30-S initiation complex joins with the 50-S subunit, IF-3 forms no part of this complex [22]. We noticed that thiostrepton seems to interfere with the coupling of both subunits and in order to clarify some of the points mentioned we decided to analyse the involvement of these components by means of sucrose gradient analysis.…”

“…As expected the inhibition is independent of the IF-2 concentration used and follows the pattern already shown for the puromycin reaction. The increase of the inhibition by thiostrepton with higher amounts of IF-3 was surprising, because this factor is released during the assembly of the 30-S initiation complex [22]. In order to clarify this point we studied the rate of exchange of Met-tRNA in 30-S initiation complexes, which were formed for 20 min at 37 "C with f[3H]Met-tRNA in presence of low and high amounts of IF-3.…”

Recognition of Initiation Codons in Modified f2 RNA by Escherichia coli Ribosomes

“…It is further substantiated by the separation of the E.coli factor IF-3 into subfractions differing in their activity towards different cistrons in mRNAs, as well as isolation of 'interference' proteins modulating the specificity of this factor [8,9]. However, binding of synthetic mRNAs, such as poly(A,U,G), poly(U) [10,11] as well as formaldehyde-treated MS2 RNA to E.coli 70-S ribosomes or 3 0 3 subunits proceeds in the absence of initiation factors [12]. On the other hand, addition of IF-1, IF-2 and IF-3 to a system containing poly(A, U, G) and 3 0 4 subunits considerably enhances binding of f!Met-tRNA [13].…”