Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

The mechanism of action of initiation factor 3 in protein synthesis. II. Association of the 30S ribosomal protein S12 with IF-3

Dimethylsuberimidate was used to crosslink ¹⁴C-labeled chain initiation factor 3 to $\text{E. coli}$ 30S particles. The crosslinked ribosomal proteins were analyzed by dodecyl sulfate polyacrylamide gel electrophoresis, and one major radioactive aggregate was found corresponding to a molecular weight of 41,000. Ribosomal protein S12 was identified to be crosslinked to IF-3 by immunological cross-reactivity.     

Initiation factor IF-3 and the binary complex between initiation factor IF-2 and formylmethionyl-tRNA are mutually exclusive on the 30-S ribosomal subunit.

The formation of 30-S initiation complexes depends strongly on initiation factor IF-3; at molar ratios of IF-3 to 30-S ribosomes up to one a stimulation is observed, whereas at ratios higher than one, initiation complex formation declines strongly. The target of the observed inhibition of fMet-tRNA binding at high concentrations of IF-3 is the 30-S initiation complex itself. On the one hand addition of IF-3 to preformed 30-S initiation complexes leads to a release of bound fMet-tRNA which is linear with the amount of factor added, whereas no effect on isolated 70-S initiation complexes is seen. The release of fMet-tRNA from preformed 30-S initiation complexes is accompanied by a release of IF-2 in a one-to-one molar ratio which is in agreement with our previous findings showing that binding of fMet-tRNA takes place via a binary complex: IF-2 . fMet-tRNA (Eur. J. Biochem. 66, 181--192 and 77, 69--75). On the other hand increasing amounts of both IF-2 and fMet-tRNA relieve the IF-3-induced inhibition of 30-S initiation complex formation. From these findings it is concluded that IF-3 and the IF-2 . fMet-tRNA complex are mutually exclusive on the 30-S ribosome. This implies that under our experimental conditions MS2 RNA binding precedes fMet-tRNA binding if one accepts that the presence of IF-3 on the 30-S subunit is obligatory for messenger binding.     

Nature of the ribosomal binding site for initiation factor 3 (IF-3)

$\text{In vitro}$ labelled IF-3 binds to both 16S and 23S rRNA but while one molecule of IF-3 binds to each 30S particle, binding to 50S particles is negligible. If proteins are removed by LiCl or CsCl treatment from either ribosomal subunit, however, binding specificity is lost and new “binding sites” appear on both ribosomal particles. Controlled RNase digestion of the 30S subunits does not cause the loss of any r-protein while controlled trypsin digestion results in the loss or degradation of several r-proteins; compared to the Phe-tRNA binding site, the binding site of IF-3 seems to be more sensitive to RNase than to trypsin digestion. Antibodies against single 30S r-proteins, which inhibit other ribosomal functions, do not prevent the binding of IF-3. RNA-binding dyes (acridine orange and pyronine) inhibit the binding of IF-3 to 30S ribosomal subunits. It is proposed that a segment of the 16S rRNA provides the binding site for IF-3 and that r-proteins confer specificity, restricting the number of available “binding sites”, and stabilize the 30S-IF-3 interaction.     

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.     

Participation of initiation factor IF-3 in the binding of AcPhe-tRNA to the 30S ribosomal subunit

Initiation factor IF-3 is required in addition to IF-1 and IF-2 for maximal initial rate of poly(U)-directed binding of AcPhe-tRNA to 30S ribosomal subunits of $\text{E. coli}$. Incubation periods longer than 10 sec, by which time the reaction is virtually over, progressively obscure the requirement for IF-3 in AcPhe-tRNA binding. IF-3 also stimulates the poly(A, G, U)-directed binding of fMet-tRNA to the 30S ribosomal subunit, but in this case, significant stimulation can still be observed even with extended incubation. These results indicate that IF-3 functions similarly in the translation of synthetic mRNA, as it does with natural mRNA, participating in ribosome dissociation and in the formation of the initiation complex from the 30S ribosomal subunit.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

The involvement of a complex between formylmethionyl-tRNA and initiation factor IF-2 in prokaryotic initiation.

A complex between initiation factor IF-2 and fMet-tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde. The IF-2 - FMet-tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di of triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF-2 - fMet-tRNA complex formation is not influenced by initiation factors IF-1 and IF-3. The binary complex can be bound to the 30-S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF-2 - fMet-tRNA complex and the nucleotide moiety to the 30-S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF-2 - fMet-tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate had no effect.     

Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties

Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond. IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes. According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed [Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J. W. B., Hansen, H. F., Petersen, H. U., Clark, B. F. C., Kjeldgaard, M., La Cour, T. F. M., Mortensen, K. K., & Nyborg, J. (1987) Biochemistry 26, 5070-5076]. A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ. The N- and C-terminal sequences of this IF-2 peptide were characterized. Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels. This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain. However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes. It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.     

Yeast ribosomal protein L12 is a substrate of protein-arginine methyltransferase 2

Type III protein-arginine methyltransferase from the yeast Saccharomyces cerevisiae (RMT2) was expressed in Escherichia coli and purified to apparent homogeneity. The cytosolic, ribosomal, and ribosome salt wash fractions from yeast cells lacking RMT2 were used as substrates for the recombinant RMT2. Using S-adenosyl-l-methionine as co-substrate, RMT2 methylated a protein in the ribosome salt wash fraction. The same protein in the ribosomal fraction was also methylated by RMT2 after pretreating the sample with endonuclease. Amino acid analysis affirmed that the labeling products were delta-N-monomethylarginines. The methylated protein from the ribosomal or the ribosome salt wash fraction was isolated by two-dimensional gel electrophoresis and identified as ribosomal protein L12 by mass spectrometry. Using synthetic peptides, recombinant L12, and its mutant as substrates, we pinpointed Arg(67) on ribosomal protein L12 as the methyl acceptor. L12 was isolated from wild type yeast cells that have been grown in the presence of S-adenosyl-l-[methyl-(3)H]methionine and subjected to amino acid analysis. The results indicate that L12 contains delta-N-monomethylarginines.