In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling.
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We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2. (+info)
Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2.
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Binding of initiator methionyl-tRNA to ribosomes is catalyzed in prokaryotes by initiation factor (IF) IF2 and in eukaryotes by eIF2. The discovery of both IF2 and eIF2 homologs in yeast and archaea suggested that these microbes possess an evolutionarily intermediate protein synthesis apparatus. We describe the identification of a human IF2 homolog, and we demonstrate by using in vivo and in vitro assays that human IF2 functions as a translation factor. In addition, we show that archaea IF2 can substitute for its yeast homolog both in vivo and in vitro. We propose a universally conserved function for IF2 in facilitating the proper binding of initiator methionyl-tRNA to the ribosomal P site. (+info)
Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.
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The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans. (+info)
The fMet-tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues.
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Mutations of the cysteines (positions 668 and 714) were generated in the IF2 C domain of Bacillus stearothermophilus translation initiation factor IF2. The corresponding proteins were characterized functionally and structurally. Most (yet not all) amino acid replacements at both positions resulted in severe reduction of the fMet-tRNA binding activity of IF2 C without grossly altering its structure. Our work demonstrates that: (a) both Cys residues are buried within an hydrophobic core and not accessible to protonation or chemical substitution, (b) neither Cys is functionally essential and (c) both Cys residues are located near the active site, probably without participating directly in fMet-tRNA binding. (+info)
Identification of Enterobacteriaceae by partial sequencing of the gene encoding translation initiation factor 2.
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Nucleotide sequence analysis is increasingly being used to identify bacteria. In this work, a PCR assay based on degenerate primers was used to obtain the partial sequence of infB, the gene encoding translation initiation factor 2 (IF2), in 39 clinical isolates of different Enterobacteriaceae. The partial sequence encodes the GTP-binding domain of IF2. Together with sequences from the literature, a total of 15 species, each represented by one to seven strains, was investigated. Phylogenetic analysis yielded an evolutionary tree which had a topology similar to a tree constructed using available 16S rRNA sequences. It is concluded that the inter-species variation of the infB gene fragment is sufficient for its use in the characterization of strains that have aberrant phenotypic reactions. (+info)
Chaperone properties of bacterial elongation factor EF-G and initiation factor IF2.
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Elongation factor G(EF-G) and initiation factor 2 (IF2) are involved in the translocation of ribosomes on mRNA and in the binding of initiator tRNA to the 30 S ribosomal subunit, respectively. Here we report that the Escherichia coli EF-G and IF2 interact with unfolded and denatured proteins, as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-G and IF2 promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with unfolded proteins such as reduced carboxymethyl alpha-lactalbumin. Furthermore, the EF-G and IF2-dependent renaturations of citrate synthase are stimulated by GTP, and the GTPase activity of EF-G and IF2 is stimulated by the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. The concentrations at which these chaperone-like functions occur are lower than the cellular concentrations of EF-G and IF2. These results suggest that EF-G and IF2, in addition to their role in translation, might be implicated in protein folding and protection from stress. (+info)
The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2.
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Previous protein unfolding studies had suggested that IF2 C, the 24. 5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule. Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-1) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721. Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain. Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids. IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry. The results indicate that IF2 C-2 is a globular molecule containing predominantly beta structures (25% antiparallel and 8% parallel beta strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1. (+info)
The fate of the initiator tRNAs is sensitive to the critical balance between interacting proteins.
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Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms. (+info)