RNA chaperone activity of translation initiation factor IF1

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.     

Ribosomal localization of translation initiation factor IF2

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.     

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

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.     

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.     

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.     

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

Solution structure of the C1-subdomain of Bacillus stearothermophilus translation initiation factor IF2

IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNAf(Met). After these interactions, fMet-tRNAf(Met) is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNAf(Met), contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNAf(Met) were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel beta-sheet flanked by three alpha-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.     

Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.     

Expression of translation initiation factor IF2 is regulated during osteoblast differentiation

We isolated and characterized a cDNA for the N-terminal half of the eukaryotic initiation of translation factor 2 (cIF2) during a screen of chicken osteoblast cDNAs. The apparent size of the message for this protein, approximately 5.6 kb, is slightly larger in size than that for human IF2 (hIF2). There is a high degree of sequence similarity between the human and chicken N-terminal portions of the protein that extends to the encoding nucleotide sequence. The tissue specific expression pattern for cIF2 and hIF2 are similar, being moderately abundant in brain, liver, and skeletal muscle, and detectable in kidney, chondrocytes, and freshly isolated osteoblasts. The ratio of message for cIF2 to that of beta-actin was 0.10 and 0.18 for liver and brain. Message levels peak in osteoblasts between 8 and 12 days of culture, coinciding with high levels of matrix protein synthesis. At peak expression, the ratio of cIF2:beta-actin for 8 day osteoblasts was 0.76. Treatment of osteoblast cultures with cycloheximide markedly reduces the level of cIF2 message indicating that novel protein synthesis is required for its expression. Hybridization of RNA samples from either chicken osteoblasts or a human osteoblast cell line with a probe for a subunit of human eukaryotic initiation of translation factor 2 (eIF2alpha), the housekeeping initiation factor, indicates that levels of eIF2 remain low. With hIF2, cIF2 represents the only other vertebrate homolog of IF2 for which a major portion of the coding sequence has been identified. This is the first report of regulated expression for a eukaryotic IF2 and is the first demonstration of its abundance in osteoblasts.     

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.     

Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.     

Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli.

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA+ gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1.     

Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome

Protein biosynthesis in bacteria is controlled by a number of translation factors. Recent data based on comparison of sequence and structure data of translation factors have established a novel hypothesis for their interaction with the ribosome: initiation, elongation, and termination factors may use a common or partly overlapping binding site on the ribosome in a process of macromolecular mimicry of an A-site-bound tRNA. This paper reviews structural knowledge and tRNA macromolecular mimicry involvement of translation initiation factor IF2. Furthermore, a model is proposed for the factor and its interaction with the ribosome during the formation of the translation initiation complex.     

Ribosome-independent GTPase activity of translation initiation factor IF2 and of its G-domain.

In the absence of ribosomes, Bacillus stearothermophilus translation initiation factor IF2 (Mr = 82 kDa) and its GTP-binding domain (i.e. the G-domain, Mr = 41 kDa) promote barely detectable hydrolysis of GTP. Upon addition of some aliphatic alcohols, however, the rate of nucleotide cleavage is substantially increased with both IF2 and G-domain, the highest stimulation being observed with 20% (v/v) ethanol. Under these conditions, the rates of ribosome-independent GTP hydrolysis with both IF2 and G-domain are approximately 30-fold lower than the corresponding rates obtained in the presence of ribosomes, while the Km for GTP is approximately the same in all cases. These results indicate that, as with the other two prokaryotic G proteins involved in translation (i.e. elongation factors EF-Tu and EF-G), also in the case of IF2, the GTPase catalytic center resides in the factor and, more specifically, in its G-domain.     

The interdomain linker of Escherichia coli initiation factor IF3: a possible trigger of translation initiation specificity

Initiation factor IF3 is responsible for the accuracy of translation initiation in bacteria, by destabilizing complexes involving non-initiator tRNA and/or nonstart codons. This proofreading is performed on the 30S subunit to which IF3 binds selectively. IF3 has an unusual architecture, with two globular domains connected by a mobile, positively charged linker. Here, we have investigated the function of this flexible tether by probing its conformation when IF3 is bound to the ribosomal RNA. Using site-directed mutagenesis of the linker region, we have also selectively modified its length, its flexibility and its chemical composition. The function of the mutant genes was assayed in vivo, and the structural and biochemical properties of some of the corresponding variant proteins were characterized in vitro. The two isolated domains of IF3 were also co-expressed in order to test the requirement for their covalent attachment. The results indicate that the physical link between the two domains of IF3 is essential for the function of this protein, but that the exact length and chemical composition of the linker can be varied to a large extent. A model is presented in which the extended linker would act as a 'strap', triggering a conformational change in the 30S subunit, which would then ensure initiator tRNA selection.     

A conserved structural motif at the N terminus of bacterial translation initiation factor IF2

The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short beta-strands and three alpha-helices, folded to form a betaalphaalphabetabetaalpha motif with the three helices packed on the same side of a small twisted beta-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51-97 are less conserved and do not appear to form a regular structure, whereas residues 98-157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2.