Ribosomal selection of mRNAs with degenerate initiation triplets.

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present.

Functional attributes of evolutionary conserved Arg45 of Wolbachia (Brugia malayi) translation initiation factor-1.

AIM: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia's novel proteins/enzymes is necessary for the development of adulticidal drugs. MATERIALS & METHODS: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. RESULTS: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. CONCLUSION: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.

Recombinant translation initiation factor-1 of Wolbachia is an immunogenic excretory secretory protein that elicits Th2 mediated immune protection against Brugia malayi.

Wolbachia, the intracellular alpha-proteobacteria are required for the development, fertility and survival of filarial parasites. Wolbachia Translation initiation factor-1 (Wol Tl IF-1) is one of the factors required for Wolbachia growth and viability. In the present study, we cloned, over expressed and purified Wol Tl IF-1 that exhibited strong immuno-reactivity with various categories of bancroftian sera. Immunization with the recombinant protein resulted into significant reduction in microfilarial density (70-72%) and adult worm establishment (61-63%) in susceptible Mastomys coucha. Protection offered by Wol Tl IF-1 was found associated with humoral immune arm as observed by an increased antibody level with preponderance of IgE, IgM, IgG1 and IgG2a isotypes. The anti-Wol Tl IF-1 antibodies promoted profound adherence of peritoneal exudates cells to the surface of microfilariae and infective larvae causing cytotoxicity and their death. The present study indicates potential of recombinant Wol Tl IF-1 as a promising vaccine candidate against human lymphatic filarial infection.

Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association.

A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.

Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region.

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

Functional investigation of residue G791 of Escherichia coli 16S rRNA: implication of initiation factor 1 in the restoration of P-site function.

Using a specialized ribosome system, previous studies have identified G791 in Escherichia coli 16S rRNA as an invariant and essential residue for ribosome function. To investigate the functional role of G791, we searched for multicopy suppressors that partially restored the protein synthesis ability of mutant ribosomes bearing a G to U substitution at position 791 (U791 ribosomes). Analyses of isolated multicopy suppressors showed that overexpression of initiation factor 1 (IF1) enhanced the protein synthesis ability of U791 ribosomes. In contrast, overexpression of initiation factor 2 (IF2) or IF3 did not enhance the protein synthesis ability of wild-type or U791 ribosomes, and overexpression of IF1 did not affect the function of wild-type or mutant ribosomes bearing nucleotide substitutions in other regions of 16S rRNA. Analyses of sucrose gradient profiles of ribosomes showed that overexpression of IF1 marginally enhanced the subunit association of U791 ribosomes and indicated lower binding affinity of U791 ribosomes to IF1. Our findings suggest the involvement of IF1 in the restoration of the P-site function that was impaired by a nucleotide substitution at residue G791.

Comparative analysis of changes in gene expression due to RNA melting activities of translation initiation factor IF1 and a cold shock protein of the CspA family.

In Escherichia coli, temperature downshift elicits cold shock response, which is characterized by induction of cold shock proteins. CspA, the major cold shock protein of E. coli, helps cells to acclimatize to low temperature by melting the secondary structures in nucleic acids and acting as a transcription antiterminator. CspA and its homologues contain the cold shock domain and belong to the oligomer binding protein family, which also includes S1 domain proteins such as IF1. Structural similarity between IF1 and CspA homologues suggested a functional overlap between these proteins. Indeed IF1 can melt secondary structures in RNA and acts as transcription antiterminator in vivo and in vitro. Here, we show that in spite of having these critical activities, IF1 does not complement cold-sensitivity of a csp quadruple deletion strain. DNA microarray analysis shows that overproduction of IF1 and Csp leads to changes in expression of different sets of genes. Importantly, several genes which were previously shown to require Csp proteins for their expression at low temperature did not respond to IF1. Moreover, in vitro, we show that a transcription terminator responsive to Csp does not respond to IF1. Our results suggest that Csp proteins and IF1 have different sets of target genes as they may be suppressing the function of different types of transcription termination elements in specific genes.

Characterization of Bacillus stearothermophilus infA and of its product IF1.

Bacillus stearothermophilus infA encoding translation initiation factor IF1 was cloned and expressed in Escherichia coli and its transcript and protein product characterized. Although the functional properties of B. stearothermophilus and E. coli IF1, compared in several translational tests in the presence of both homologous and heterologous components, are not entirely identical, the two proteins are interchangeable in an in vitro translational system programmed with a natural mRNA. The availability of purified B. stearothermophilus IF1 now allows us to analyze the translation initiation pathway using efficient in vitro tests based entirely on purified components derived from this thermophilic Gram-positive bacterium.

Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1-IF2 interaction.

Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1-IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S-IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2DeltaN, regardless of 30S subunits and IF2 origin. We conclude that an IF1-IF2 interaction is not universally conserved and is not essential for cell survival.

Structure of the 30S translation initiation complex.

Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.

Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis.

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.

A single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors.

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

Production and epitope characterization of mAbs specific for translation factor IF1.

Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone. Despite extensive research, the exact function of IF1 in translation initiation has not yet been determined, and the research in the function of the factor has in some areas been impeded by the lack of monoclonal antibodies specific for this protein. Several attempts to induce immune response in mice with wild-type IF1 for the production of antibodies have failed. We have now succeeded in producing monoclonal antibodies specific for IF1 by applying a new immunization strategy involving an antigen combination of IF1 coupled to glutathione S-transferase (GST) and a recombinant dimer of IF1. This resulted in the generation of 6 IgG, 2 IgM, and 1 IgA anti-IF1 antibodies, which can be used in ELISA screening and Western immunoblots. We also provide a mapping of the functional epitopes of the generated anti-IF1 monoclonal antibodies by screening the antibodies for binding to IF1 proteins mutated at single amino acid positions.

Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation.

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37 degrees C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold-shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of initiation factor 1 from Mycobacterium tuberculosis.

Initiation factor 1 (IF-1; Rv3462c) from Mycobacterium tuberculosis, a component of the 30S initiation complex, was cloned and heterologously expressed in Escherichia coli. The protein was purified by affinity and size-exclusion chromatography and crystallized. A complete data set has been collected to high resolution. The crystals belonged to space group P2(1)2(1)2, with two molecules per asymmetric unit which are related by translational symmetry.

Transcription antitermination by translation initiation factor IF1.

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of Escherichia coli) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the E. coli translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.

Differential promoter usage of infA in response to cold shock in Escherichia coli.

Initiation factor 1 (IF1) is an essential protein in Escherichia coli involved in the initiation step of protein synthesis. The protein level of IF1 increases when E. coli cells are subjected to cold shock, however, it remains unclear as to how this increase occurs. The infA gene encoding IF1 contains two promoters, the distal P1 and the proximal P2 promoter. In this study, we found that infA mRNA was greatly increased, and that this increase resulted from transcriptional activation of P1, not P2, during cold shock although stability of transcripts from both promoters concomitantly increased.

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level.

The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A' reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.

A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli.

Uneven distribution of plasmid-based expression vectors to daughter cells during bacterial cell division results in an increasing proportion of plasmid free cells during growth. This is a major industrial problem leading to reduction of product yields and increased production costs during large-scale cultivation of vector-carrying bacteria. For this reason, a selection must be provided that kills the plasmid free cells. The most conventional method to obtain this desired selection is to insert some gene for antibiotic resistance in the plasmid and then grow the bacteria in the presence of the corresponding antibiotic. We describe here a host/plasmid Escherichia coli system with a totally stable plasmid that can be maintained without the use of antibiotic selection. The plasmid is maintained, since it carries the small essential gene infA (coding for translation initiation factor 1, IF1) in an E. coli strain that has been deleted for its chromosomal infA gene. As a result only plasmid carrying cells can grow, making the strain totally dependent on the maintenance of the plasmid. A selection based on antibiotics is thus not necessary during cultivation, and no antibiotic-resistance genes are present neither in the final strain nor in the final plasmid. Plasmid-free cells do not accumulate even after an extended period of continuous growth. Growth rates of the control and the plasmid harboring strains are indistinguishable from each other in both LB and defined media. The indicated approach can be used to modify existing production strains and plasmids to the described concept. The infA based plasmid stability system should eliminate industrial cultivation problems caused by the loss of expression vector and use of antibiotics in the cultivation medium. Also environmental problems caused by release of antibiotics and antibiotic resistance genes, that potentially can give horizontal gene transfer between bacterial populations, are eliminated.