The dynamic cycle of bacterial translation initiation factor IF3.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

1H, 13C and 15N resonance assignments of translation initiation factor 3 from Pseudomonas aeruginosa.

Translation initiation factor 3 (IF3) is one of the three protein factors that bind to the small ribosomal subunit and it is required for the initiation of protein biosynthesis in bacteria. IF3 contains two independent domains, N- and C-terminal domains, which are connected by a lysine-rich interdomain linker. IF3 undergoes large-scale movements and conformational changes upon binding to the 30S subunit and also during the functional regulation of initiation. However, the precise dynamic interplay of the two domains and the molecular mechanism of IF3 is not well understood. A high-resolution 3D structure of a complete IF3 in bacteria has not been solved. Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is a primary cause of nosocomial infections in humans. Here we report the NMR chemical shift assignments of IF3 from P. aeruginosa as the first step toward NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data identified nine ß-strands and four α-helices arranged in the sequential order ß1-ß2-α1-ß3-ß4-α2-ß5-α3-ß6-α4-ß7-ß8-ß9.

Biological and Evolutionary Significance of Terminal Extensions of Mitochondrial Translation Initiation Factor 3.

Protein biosynthesis in mitochondria is organized in a bacterial manner. However, during evolution, mitochondrial translation mechanisms underwent many organelle-specific changes. In particular, almost all mitochondrial translation factors, being orthologous to bacterial proteins, are characterized by some unique elements of primary or secondary structure. In the case of the organellar initiation factor 3 (IF3), these elements are several dozen amino acids long N- and C-terminal extensions. This study focused on the terminal extensions of baker's yeast mitochondrial IF3, Aim23p. By in vivo deletion and complementation analysis, we show that at least one extension is necessary for Aim23p function. At the same time, human mitochondrial IF3 is fully functional in yeast mitochondria even without both terminal extensions. While Escherichia coli IF3 itself is poorly active in yeast mitochondria, adding Aim23p terminal extensions makes the resulting chimeric protein as functional as the cognate factor. Our results show that the terminal extensions of IF3 have evolved as the "adaptors" that accommodate the translation factor of bacterial origin to the evolutionary changed protein biosynthesis system in mitochondria.

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits.

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition.

Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.

Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

Architecture of human translation initiation factor 3.

Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans.

Conformational selection of translation initiation factor 3 signals proper substrate selection.

During translation, initiation factor 3 (IF3) binds to the small (30S) ribosomal subunit and regulates the fidelity with which the initiator tRNA and mRNA start codon substrates are selected into the 30S initiation complex (30S IC). The molecular mechanism through which IF3 promotes the recognition and signaling of correct substrate selection, however, remains poorly defined. Using single-molecule fluorescence resonance energy transfer, we show that 30S IC-bound Escherichia coli IF3 exists in a dynamic equilibrium between at least three conformations. We found that recognition of a proper anticodon-codon interaction between initiator tRNA and the start codon within a completely assembled 30S IC selectively shifts this equilibrium toward a single conformation of IF3. Our results strongly support a conformational selection model in which the conformation of IF3 that is selectively stabilized within a completely and correctly assembled 30S IC facilitates further progress along the initiation pathway.

Real-time assembly landscape of bacterial 30S translation initiation complex.

Initiation factors guide the ribosome in the selection of mRNA and translational reading frame. We determined the kinetically favored assembly pathway of the 30S preinitiation complex (30S PIC), an early intermediate in 30S initiation complex formation in Escherichia coli. IF3 and IF2 are the first factors to arrive, forming an unstable 30S-IF2-IF3 complex. Subsequently, IF1 joins and locks the factors in a kinetically stable 30S PIC to which fMet-tRNA(fMet) is recruited. Binding of mRNA is independent of initiation factors and can take place at any time during 30S PIC assembly, depending on the cellular concentration of the mRNA and the structural determinants at the ribosome-binding site. The kinetic analysis shows both specific and cumulative effects of initiation factors as well as kinetic checkpoints of mRNA selection at the entry into translation.

Interaction of IF2 with the ribosomal GTPase-associated center during 70S initiation complex formation.

Addition of an Escherichia coli 50S subunit (50S(Cy5)) containing a Cy5-labeled L11 N-terminal domain (L11-NTD) within the GTPase-associated center (GAC) to an E. coli 30S initiation complex (30SIC(Cy3)) containing Cy3-labeled initiation factor 2 complexed with GTP leads to rapid development of a FRET signal during formation of the 70S initiation complex (70SIC). Initiation factor 2 (IF2) and elongation factor G (EF-G) induce similar changes in ribosome structure. Here we show that such similarities are maintained on a dynamic level as well. Thus, movement of IF2 toward L11-NTD after initial 70S ribosome formation follows GTP hydrolysis and precedes P(i) release, paralleling movement of EF-G following its binding to the ribosome [Seo, H., et al. (2006) Biochemistry 45, 2504-2514], and in both cases, the rate of such movement is slowed if GTP hydrolysis is prevented. The 30SIC(Cy3):50S(Cy5) FRET signal also provides a sensitive probe of the ability of initiation factor 3 to discriminate between a canonical and a noncanonical initiation codon during 70SIC formation. We employ Bacillus stearothermophilus IF2 as a substitute for E. coli IF2 to take advantage of the higher stability of the complexes it forms with E. coli ribosomes. While Bst-IF2 is fully functional in formation of E. coli 70SIC, relative reactivities toward dipeptide formation of 70SICs formed with the two IF2s suggest that the Bst-IF2.GDP complex is more difficult to displace from the GAC than the E. coli IF2.GDP complex.

Evidence for an active role of IF3mt in the initiation of translation in mammalian mitochondria.

Mitochondrial translational initiation factor 3 (IF3(mt)) is a 29 kDa protein that has N- and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3(mt) important for its activity. On the basis of homology models of IF3(mt), mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170-171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with K(d) values similar to that of the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3(mt) to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3(mt) to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3(mt) plays an active role in initiation of translation.

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.

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 approximately 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 muM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K(d) = 0.12-0.25 muM). 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 real-time path of translation factor IF3 onto and off the ribosome.

Translation initiation factor IF3 is an essential bacterial protein, consisting of two domains (IF3C and IF3N) separated by a linker, which interferes with ribosomal subunit association, promotes codon-anticodon interaction in the P site, and ensures translation initiation fidelity. Using time-resolved chemical probing, we followed the dynamic binding path of IF3 on the 30S subunit and its release upon 30S-50S association. During binding, IF3 first contacts the platform (near G700) of the 30S subunit with the C domain and then the P-decoding region (near A790) with its N domain. At equilibrium, attained within less than a second, both sites are protected, but before reaching binding equilibrium, IF3 causes additional transient perturbations of both the platform edge and the solvent side of the subunit. Upon 30S-50S association, IF3 dissociates concomitantly with the establishment of the 30S-50S bridges, following the reverse path of its binding with the IF3N-A790 interaction being lost before the IF3C-G700 interaction.

Mechanism of recycling of post-termination ribosomal complexes in eubacteria: a new role of initiation factor 3.

Ribosome recycling is a process which dissociates the post-termination complexes (post-TC) consisting of mRNA-bound ribosomes harbouring deacylated tRNA(s). Ribosome recycling factor (RRF), and elongation factor G (EFG) participate in this crucial process to free the ribosomal subunits for a new round of translation. We discuss the over-all pathway of ribosome recycling in eubacteria with especial reference to the important role of the initiation factor 3 (IF3) in this process. Depending on the step(s) at which IF3 function is implicated, three models have been proposed. In model 1, RRF and EFG dissociate the post-TCs into the 50S and 30S subunits, mRNA and tRNA(s). In this model, IF3, which binds to the 30S subunit, merely keeps the dissociated subunits apart by its anti-association activity. In model 2, RRF and EFG separate the 50S subunit from the post-TC. IF3 then dissociates the remaining complex of mRNA, tRNA and the 30S subunit, and keeps the ribosomal subunits apart from each other. However, in model 3, both the genetic and biochemical evidence support a more active role for IF3 even at the step of dissociation of the post-TC by RRF and EFG into the 50S and 30S subunits.

The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH.

Eukaryotic initiation factor eIF1 and the functional C-terminal domain of prokaryotic initiation factor IF3 maintain the fidelity of initiation codon selection in eukaryotes and prokaryotes, respectively, and bind to the same regions of small ribosomal subunits, between the platform and initiator tRNA. Here we report that these nonhomologous factors can bind to the same regions of heterologous subunits and perform their functions in heterologous systems in a reciprocal manner, discriminating against the formation of initiation complexes containing codon-anticodon mismatches. We also show that like IF3, eIF1 can influence initiator tRNA selection, which occurs at the stage of ribosomal subunit joining after eIF5-induced hydrolysis of eIF2-bound GTP. The mechanisms of initiation codon and initiator tRNA selection in prokaryotes and eukaryotes are therefore unexpectedly conserved and likely involve related conformational changes induced in the small ribosomal subunit by factor binding. YciH, a prokaryotic eIF1 homologue, could perform some of IF3's functions, which justifies the possibility that YciH and eIF1 might have a common evolutionary origin as initiation factors, and that IF3 functionally replaced YciH in prokaryotes.

Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection.

The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui(-) phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui(-) phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui(-) phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.

Mapping the active sites of bacterial translation initiation factor IF3.

IF3C is the C-terminal domain of Escherichia coli translation initiation factor 3 (IF3) and is responsible for all functions of this translation initiation factor but for its ribosomal recycling. To map the number and nature of the active sites of IF3 and to identify the essential Arg residue(s) chemically modified with 2,3-butanedione, the eight arginine residues of IF3C were substituted by Lys, His, Ser and Leu, generating 32 variants that were tested in vitro for all known IF3 activities. The IF3-30S subunit interaction was inhibited strongly by substitutions of Arg99, Arg112, Arg116, Arg147 and Arg168, the positive charges being important at positions 116 and 147. The 70S ribosome dissociation was affected by mutations of Arg112, Arg147 and, to a lesser extent, of Arg99 and Arg116. Pseudo-initiation complex dissociation was impaired by substitution of Arg99 and Arg112 (whose positive charges are important) and, to a lesser extent, of Arg116, Arg129, Arg133 and Arg147, while the dissociation of non-canonical 30S initiation complexes was preserved at wild-type levels in all 32 mutants. Stimulation of mRNA translation was reduced by mutations of Arg116, Arg129 and, to a lesser extent, of Arg99, Arg112 and Arg131 whereas inhibition of non-canonical mRNA translation was affected by substitutions of Arg99, Arg112, Arg168 and, to a lesser extent, Arg116, Arg129 and Arg131. Finally, repositioning the mRNA on the 30S subunit was affected weakly by mutations of Arg133, Arg131, Arg168, Arg147 and Arg129. Overall, the results define two active surfaces in IF3C, and indicate that the different functions of IF3 rely on different molecular mechanisms involving separate active sites.

Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3.

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.

Solution structure of the R3H domain from human Smubp-2.

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy. It is the first 3D structure determination of an R3H domain. The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3. The similarities are non-trivial, as the amino acid identities are below 10%. Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition. An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site. A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.