Roles of the N- and C-terminal domains of mammalian mitochondrial initiation factor 3 in protein biosynthesis

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3_mt) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3_mt with and without the connecting linker were prepared. The K_d values for the binding of full-length IF3_mt and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3_mt. This observation indicates that the N-domain of IF3_mt has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3_mt for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3_mt in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3_mt reduces the binding of fMet-tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3_mt promotes the formation of a binary complex between IF2_mt and fMet-tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.     

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt

Mammalian mitochondrial initiation factor 3 (IF3_mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3_mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K_d for the binding of IF3_mt to 28S subunits is ~30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3_mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K_d = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K_d = 0.12−0.25 μM). IF3_mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3_mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3_mt from the 28S subunits upon 39S subunit joining.     

The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit

Bovine mitochondrial translational initiation factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.     

Structural dynamics of bacterial translation initiation factor IF2

Bacterial translation initiation factor IF2 promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site and initiation dipeptide formation. Here, we present the solution structures of GDP-bound and apo-IF2-G2 of Bacillus stearothermophilus and provide evidence that this isolated domain binds the 50 S ribosomal subunit and hydrolyzes GTP. Differences between the free and GDP-bound structures of IF2-G2 suggest that domain reorganization within the G2-G3-C1 regions underlies the different structural requirements of IF2 during the initiation process. However, these structural signals are unlikely forwarded from IF2-G2 to the C-terminal fMet-tRNA binding domain (IF2-C2) because the connected IF2-C1 and IF2-C2 modules show completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity that was found in the archaeal IF2 homolog aIF5B.     

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.     

Computational exploration of structural hypotheses for an additional sequence in a mammalian mitochondrial protein

Background

Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2_mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2_mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.

Results

The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2_mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2_mt predictions differ from the observed IF2_mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.

Conclusions

By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2_mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2_mt cryo-EM map to get reasonable quasi-atomic IF2_mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2_mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.     

The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.     

A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3_Y75N protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.     

Relationship between size of mRNA ribosomal binding site and initiation factor function

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.     

A quantitative kinetic scheme for 70 S translation initiation complex formation

Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50 S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and fMet-tRNA(f)(Met). According to this scheme, an initially formed labile 70 S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30 S and 50 S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA(fMet), which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA(fMet). The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the non-hydrolyzable analog GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70 S formation, but perturbs fMet-tRNA(fMet) positioning in the P-site. In contrast, thiostrepton severely retards stable 70 S formation, but allows normal binding of fMet-tRNA(fMet)(prf20) to the P-site.     

Initiation of protein synthesis in animal mitochondria. Purification and characterization of translational initiation factor 2

Bovine liver mitochondrial translational initiation factor 2 (IF-2mt) has been purified to near homogeneity. The scheme developed results in a 24,000-fold purification of the factor with about 26% recovery of activity. SDS-polyacrylamide gel electrophoresis indicates that IF-2mt has a subunit molecular mass of 85 kDa. IF-2mt promotes the binding of formyl(f)Met-tRNA to mitochondrial ribosomes but is inactive with the nonformylated derivative. IF-2mt is active on chloroplast 30 S ribosomal subunits, but IF-2chl has no activity in promoting fMet-tRNA binding to animal mitochondrial ribosomes. IF-2mt is sensitive to elevated temperatures and is inactivated by treatment with N-ethylmaleimide. It is partially protected from heat and N-ethylmaleimide inactivation by the presence of either GTP or GDP suggesting that guanine nucleotides may bind to this factor directly. The binding of fMet-tRNA to mitochondrial ribosomes requires the presence of GTP and is inhibited by GDP. DeoxyGTP is very effective in replacing GTP in promoting fMet-tRNA binding to ribosomes and some activity is also observed with ITP. No activity is observed with ATP, CTP, or UTP. Nonhydrolyzable analogs of GTP can promote formation of both 28 S and 55 S initiation complexes indicating that GTP hydrolysis is not required for subunit joining in the animal mitochondrial system.     

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA _inff ^supMet . Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA _inff ^supMet . Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU₃ as mRNA, elongator-tRNAs competed with fMet-fRNA _inff ^supMet and with tRNA _inff ^supMet . fMet-tRNA _inff ^supMet reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU₃ complex at the A site only if fMet-tRNA _inff ^supMet or tRNA _inff ^supMet filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.     

Polypeptide chain initiation in eukaryotes. IV. Purification and properties of supernatant initiation factor from Artemia salina embryos

The eukaryotic supernatant initiation factor, described in earlier publications from this laboratory, has been isolated and purified over 3000-fold, to about 70 to 80% purity, from extracts of embryos of the brine shrimp Artemia salina. The native protein appears to consist of two equal subunits, each weighing approximately 74,000 daltons. Like the bacterial initiation factor IF2, its prokaryotic counterpart, the Artemia factor promotes the AUG-dependent binding of fMet-tRNA, or the poly (U)-dependent binding of N-acetyl-Phe-tRNA, to the small ribosomal subunit. However, unlike IF2, the reaction is GTP-independent and the factor functions catalytically for one molecule may promote the binding of up to 12 molecules of fMet-tRNA to 40 s subunits at 0 °C.     

The cryo-EM structure of a translation initiation complex from Escherichia coli

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.     

Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.