Translation Initiation Factor IF2 Research Articles

Improved Recombinant Tandem Expression of Translation Initiation Factor IF2 in RNase E Deficient E. coli Cells

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2α, IF2β and IF2γ differing only in the N-terminal: IF2β and IF2γ lack 158 and 165 amino acid residues, respectively, as compared to IF2α. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2α, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2β and IF2γ is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2. Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2α and IF2β/IF2γ. The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites. However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2α and IF2β/IF2γ is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant protein production and expression efficiency.

X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix.

The structures of the two domains of translational initiation factor IF3 from Bacillus stearothermophilus have been solved by X-ray crystallography using single wavelength anomalous scattering and multiwavelength anomalous diffraction. Each of the two domains has an alpha/beta topology, with an exposed beta-sheet that is reminiscent of several ribosomal and other RNA binding proteins. An alpha-helix that protrudes out from the body of the N-terminal domain towards the C-terminal domain suggests that IF3 consists of two RNA binding domains connected by an alpha-helix and that it may bridge two regions of the ribosome. This represents the first high resolution structural information on a translational initiation factor.

Prokaryotic Translation Initiation Factor IF3 Is an Elongated Protein Consisting of Two Crystallizable Domains

We show that translation initiation factor IF3 can be split into two fragments of nearly equal size by the Escherichia coli outer membrane protease omptin. Circular dichroism and small-angle neutron scattering show that the two fragments are structured as domains. Each domain is relatively compact, and they are separated by about 45 A in intact IF3. Thus IF3 is an elongated protein that consists of two well-separated domains. We suggest that these two domains are involved in ribosome binding across the cleft of the 30S ribosome. We also report the crystallization of each domain of IF3.

Translation initiation factor IF1 is essential for cell viability in Escherichia coli.

Translation initiation factor IF1 is a highly conserved element of the prokaryotic translational apparatus. It has been demonstrated earlier that the factor stimulates in vitro the initiation phase of protein synthesis. However, no mutation in its gene, infA, has been identified, and a role for IF1 in translation has not been demonstrated in vivo. To elucidate the function of IF1 and determine if the protein is essential for cell growth, the chromosomal copy of infA was disrupted. Cell viability is maintained only when infA is expressed in trans from a plasmid, thereby demonstrating that IF1 is essential for cell growth in Escherichia coli. Cells depleted of IF1 exhibit few polysomes, suggesting that IF1 functions in the initiation phase of protein synthesis.

In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2.

During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.

Selective amplification of DNA fragments coding for the G domain of factors IF2 and EF-Tu, two G proteins from the myxobacterium Stigmatella aurantiaca.

Two DNA fragments of the genome of the myxobacterium Stigmatella aurantiaca were selectively amplified by PCR. These fragments encode a segment of the G domain of translational initiation factor IF2 and elongation factor EF-Tu, two GTP-binding proteins. This was made possible by carefully designing the primers for this reaction to avoid the amplification of every G-domain-encoding region of the genome. The sequence of two pairs of primers was deduced from highly conserved regions, namely G1 and G3 and/or their vicinal amino acids, within each subfamily (initiation and elongation factors, respectively) of GTP-binding proteins. On the basis of the expected size, one band was selected in each experiment, cloned into a vector, and sequenced. This showed unambiguously after comparison analysis that they belong to the IF2 and EF-Tu genes, respectively. This strategy seems suitable for the amplification of a segment of any gene coding for a G protein from any origin.

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species

Identification of a putative infC-rpml-rplT operon flanked by long inverted repeats in Mycoplasma fermentons (incognitus strain)

A specific 1542-bp DNA fragment was amplified from Mycoplasma fermentons (incognitus strain) using a unique 23-nucleotide nt) synthetic deoxyribonucleotide (oligo) (5'-TCCAAAAAGTCCGGAATTTGGGG) as the primer pair in the polymerase chain reaction (PCR). The 23-nt sequence is part of the 29-bp terminal inverted repeat (IR) which forms the left potential stem-and-loop (s&1) structure of the previously identified M. fermentans insertion-sequence(IS)-like genetic element [Hu et al., Gene 93 (1990) 67–72]. The amplified DNA was cloned and sequenced. A pair of 27-bp IR containing the 23-nt synthetic oligo was identified at both termini. Between the IR, there are four potential open reading frames (ORFs) which are arranged adjacent to each other in the order, ORF-1, ORF-2, ORF-3 and ORF-4, with parts of ORF-1 and ORF-2 overlapping. The deduced amino acid (aa) sequences of ORF-2, ORF-3 and ORF-4 are 34 to 60% identical to the translation initiation factor IF3 (encoded by the infC gene), ribosomal proteins L35 ( rpmI gene) and L20 ( rplT gene) of Escherichia coli and Bacillus stearothermophilus, respectively. In bacteria, the infC-rpml-rplT genes are organized to function as an operon. There are multiple sites with promoter-like sequences identified upstream from the putative infC gene in the mycoplasma closely resembling the gene arrangement in the bacterial operon. All three genes of ORF-2, ORF-3 and ORF-4 are preceded individually by a strong appropriately spaced (7 and 10 bp) putative Shine-Dalgarno sequence (5'-AAGGA). In addition, ORF-2 uses the unusual triplet, ATT, as the start codon, the same as that for infC in the bacterial operon. Thus, the cluster of genes (ORF-2, ORF-3 and ORF-4) is identified as a putative mycoplasma infC-rpml-rplT operon. Most interestingly, our study reveals that this operon potentially constitutes a part of a mobile genetic element in the incognitus strain of M. fermentans.

Domain of E. coli translational initiation factor IF2 homologous to lambda cI repressor and displaying DNA binding activity

The carboxy-terminal region of translational initiation factor IF2 is a common region to the three active forms of the factor (α,β and γ) but its function is still unknown. We report here that this region of IF2 carries at least one domain which is homologous to the N-terminal and middle part of the cI repressor of lambda phage. The IF2 homologous domain harbors functionally important features of the lambda represser, e.g. the helix-tum-helix motif and some of the residues essential for the structure of the hydrophobic core of the represser. This homologous domain of IF2 was fused to the β-galactosidase protein. The hybrid protein, as well as IF2 itself, shows a consistent DNA binding activity in nitrocellulose filtration assays but does not display the specificity of the cI represser for the P r operator. The implication of this domain in the transcriptional activity of IF2, reported by others, is discussed.

Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron ( rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would basepair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

Translational initiation factors IF-1 and eIF-2α share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2α chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2α may bind to RNA.

Both forms of translational initiation factor IF2 (α and β) are required for maximal growth of Escherichia coli: Evidence for two translational initiation codons for IF2β

The gene infB codes for two forms of translational initiation factor IF2; IF2α (97,300 Da) and IF2β (79,700 Da). IF2β arises from an independent translational event on a GUG codon located 471 bases downstream from IF2α start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2β synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2β results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2β have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2β exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2β start codons could we construct plasmids that express only IF2α. A plasmid expressing only IF2β was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic λ phage, we could cure the λ phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 °C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet-tRNA

Translation initiation factor IF2 from Bacillus steaiothermophilus (741 amino acids, M r 82,043) was subjected to trypsinolysis alone or in the presence of fMet-tRNA. The initiator tRNA was found to protect very efficiently the Arg 308-Ala 309 bond within the GTP binding site of IF2 and, more weakly, three bonds (Lys 146-Gln 147, Lys 154-Glu 155 and Arg 519-Ser 520). The first two are located at the border between the non-conserved, dispensable (for translation) N-terminal portion and the conserved G-domain of the protein, the third is located at the border between the G- and C-domains. Since IF2 is known to interact with fMet-tRNA through its protease-resistant C- (carboxyl terminus) domain, the observed protection suggests that, upon binding or fMet-tRNA, long-distance tertiary interactions between the IF2 domains may take place.

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.

Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule.

Structure and expression of the infA operon encoding translational initiation factor IF1. Transcriptional control by growth rate.

The cellular levels of the three translational initiation factors, IF1, IF2, and IF3, increase as a function of growth rate in parallel with those of ribosomes. Therefore both ribosomal and initiation factor gene expression is under metabolic control. To address how expression of the Escherichia coli gene for IF1, infA, is regulated, a 3-kilobase region of the genome surrounding infA was sequenced. The 5' and 3' termini of in vivo infA transcripts were defined by S1 nuclease mapping, and mRNA size was measured by Northern blot hybridization. The infA gene is transcribed by two promoters, P1 and P2, which generate transcripts of 525 and 330 nucleotides, apparently ending at the same rho-independent terminator. Analyses of operon and protein fusions to lacZ demonstrate that neither infA transcription nor translation is affected by high cellular levels of IF1. However, P2, but not P1, increases in activity as a function of the growth rate of the cell and is the dominant promoter in rich medium. Therefore, metabolic control of infA expression occurs exclusively at the level of transcription by the P2 promoter.

Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by GTP

Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, M r = 82 043) was subjected to trypsinolysis alone or in the presence of GTP. Following electroblotting and automated amino acid sequencing of the resulting peptides, the location and the sequential order of the main cleavage sites were identified. Trypsinolysis of IF2 ultimately generates two compact domains: a 24.5 kDa C-terminal fragment and a 40 kDa G-fragment which is obtained only in the presence of GTP which strongly protects a cleavage site within the GTP binding domain.

Purified internal G-domain of translational initiation factor IF-2 displays guanine nucleotide binding properties

Translational initiation factor IF-2 is involved in a multistep pathway leading to the synthesis of the first peptide bond. IF-2 is a guanine nucleotide binding protein (G-protein) and catalyzes GTP hydrolysis in the presence of ribosomes. According to sequence homologies with other G-proteins, particularly EF-Tu, a theoretical model for the tertiary structure of the putative G-domain of IF-2 has been previously proposed [Cenatiempo, Y., Deville, F., Dondon, J., Grunberg-Manago, M., Hershey, J. W. B., Hansen, H. F., Petersen, H. U., Clark, B. F. C., Kjeldgaard, M., La Cour, T. F. M., Mortensen, K. K., & Nyborg, J. (1987) Biochemistry 26, 5070-5076]. A short fragment of IF-2 encompassing the putative G-domain was purified by limited proteolysis of a chimeric protein, synthesized from a gene fusion, between a segment of the IF-2 gene and lacZ. The N- and C-terminal sequences of this IF-2 peptide were characterized. Its calculated length is 181 amino acids and its molecular mass 19.4 kDa, whereas it migrates at 14 kDa in SDS-polyacrylamide gels. This segment of IF-2 can form binary complexes with GDP and can be cross-linked to GTP, therefore indicating that it really corresponds to the G-domain. However, in contrast to the situation described for the purified G-domain of EF-Tu, the IF-2 fragment did not hydrolyze GTP even in the presence of ribosomes. It is assumed that active centers of IF-2 located outside the G-domain are needed for the latter reaction.(ABSTRACT TRUNCATED AT 250 WORDS)