Translation initiation factor IF3: two domains, five functions, one mechanism?

Abstract

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from 'stand-by' to 'P-decoding' site; (iii) dissociation of 30S-poly(U)-NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain-30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S-IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem-loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.

Fig. 1. Interaction between native IF3 and the isolated N-domain (IF3N) and C-domain (IF3C) of IF3 with 30S ribosomal subunits measured through the capacity of increasing amounts of each protein to compete with ³⁵S-labelled native IF3 for a fixed amount of 30S ribosomal subunits. Competition was quantified by incubating a fixed amount of 30S subunits and radioactive IF3 with the indicated amounts of native IF3 (filled circles), IF3C (filled triangles) and IF3N (filled squares), and measuring the amount of radioactive free protein released in the supernatant following centrifugation of the ribosomal subunits by Airfuge (Beckman). Additional experimental details can be found in Materials and methods.

Fig. 2. Dissociation of 70S ribosomes by increasing concentrations of native IF3 and IF3C. The subunit anti-association activity of the protein was measured quantitatively by preparing 70S tight couples containing ³⁵S-labelled 30S subunits and non-labelled 50S subunits. The 70S ribosomes were incubated with the amounts of either native IF3 (filled circles) or IF3C (filled triangles) indicated in the abscissa of (D) and then subjected to sucrose density gradient centrifugation to determine the amount of radioactive 30S sedimenting as 70S or as free 30S. Examples of these analyses are presented for 70S without protein added (A) or following incubation with a 20-fold stoichiometric excess of IF3 (B) or with a 200-fold excess of IF3C (C). The 70S dissociation curve obtained by the addition of increasing amounts of IF3 or IF3C is shown (D).

Fig. 3. Effect of IF3 and IF3C on the translation of two natural mRNAs as a function of their initiation codon. The reaction mixtures contained IF1 mRNA beginning with either AUG (A) or AUU (B) initiation codon, 70S ribosomes and a 1:1 stoichiometric amount of IF1 and IF2, while the amount of IF3 (filled circles) or IF3C (filled triangles) was varied as indicated in the abscissa. The IF1 product synthesized in each reaction tube was analysed by SDS–PAGE and quantified with a Molecular Imager (Bio-Rad). The results were normalized taking the level of translation obtained in the absence of either IF3 or IF3C as equal to one.

Fig. 4. Dissociation of pseudo-initiation and non-canonical initiation complexes by IF3 and IF3C. The complexes containing 30S subunits and the aminoacyl-tRNAs and templates specified below were prepared as described in Materials and methods, and then subjected to 25-fold dilution with 30 mM Tris–HCl pH 7.1, 50 mM NH₄Cl, 15 mM magnesium acetate, 2 mM 2-mercaptoethanol, containing the indicated amounts of either IF3 (filled circles) or IF3C (filled triangles). After nitrocellulose filtration, the IF3-induced dissociation of the complexes was measured through the loss of the corresponding aminoacyl-tRNA radioactivity from the filters. (A) Pseudo-initiation complex of 30S, poly(U) and NAc [¹⁴C]Phe-tRNA; (B) non-canonical initiation complex of 30S, AUU IF1 mRNA and f [³⁵S]Met-tRNA; (C) pseudo-initiation complexes of 30S, NAc [¹⁴C]Phe-tRNA and poly(U) (filled circles) or non-canonical initiation complexes of 30S, f [³⁵S]Met-tRNA and AUU IF1 mRNA (open circles). Further experimental details are given in Materials and methods.

Fig. 5. Effect of IF3 and IF3C on the mRNA shift on the 30S ribosomal subunit. (A) To illustrate the IF3-induced repositioning of the mRNA on the 30S subunit described previously (La Teana et al., 1995), we show the main site-directed mRNA–30S cross-linking sites obtained in the absence (left) and presence (right) of IF3. (B) Autoradiography of the SDS–PAGE separation of the ribosomal proteins (indicated by the arrows on the left side) cross-linked to the mRNA in initiation complexes formed, as indicated above the corresponding lanes, in the absence or presence of either IF3 or IF3C (at a 5-fold higher concentration than IF3). (C) Autoradiography of RNaseH digestion products of 16S rRNA UV cross-linked to ³²P-labelled mRNA within 30S–mRNA complexes prepared in the presence of IF1, IF2 and, where indicated, IF3 or increasing amounts of IF3C. The RNaseH digestion was carried out in the presence of oligonucleotides complementary to three regions of 16S rRNA, namely (A) 1298–1307, (B) 1382–1391 and (C) 1491–1507. Two separate digestions were performed with two different pairs of oligonucleotides, A/B (lanes marked 1) and B/C (lanes marked 2), respectively. The bands marked 45 and 150, which are obtained with all types of complexes, correspond to cross-links between –3 and +2 of mRNA and a single site (1530) of 16S rRNA, while the two additional bands marked 80 and 110, obtained exclusively from the complexes containing IF3 or IF3C, correspond to cross-links between –3 of the mRNA and 1360 of 16S rRNA, and between +11 of the mRNA and 1395 of 16S rRNA, respectively. The experimental details are identical to those described previously and it should be noted that although the electrophoretic analysis does not allow the separation of S9/S11 and S18/S21, all these proteins were demonstrated to be cross-linked to the mRNA by immunological analysis (Dontsova et al., 1991; La Teana et al., 1995).

Fig. 6. Involvement of the two IF3 domains in the modulation of IF3 binding to and release from 30S ribosomal subunits. The model rationalizes the existence of two domains interacting with two separate sites on the 30S ribosomal subunit. IF3 binds initially through its C-domain (main contact), and this allows the interaction of the N-domain with the second site (minor contact), which enhances the thermodynamic stability of the complex. The interaction with the 50S subunit induces a conformational change of the 30S subunit, which increases the distance between the two IF3 sites and causes the consequent loss of the minor contact between the subunit and the N-domain of IF3. This results in the reversal of the thermodynamic stabilization that had been provided by the 30S–N-domain interaction and favours the dissociation of IF3 from the small subunit.