Initiation Factor IF2 Research Articles

Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate

Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.

Ageing associated proteomic variations in seminal plasma of Indian men

Recent decade has seen a significant increase in the average age at which couples first produce, thus it becomes essential to study and understand how age affects fertility in humans. Age-dependent variations in human seminal plasma proteome were identified by two- dimensional differential gel electrophoresis (2D-DiGE) followed by mass spectrometric analysis. Overall, 10 differentially expressed proteins were identified, viz. glutaredoxin domain-containing cysteine-rich protein-2, clusterin, serum albumin, translation initiation factor IF-2 like, alternative protein RRT-34, ecto-ADP-ribosyltransferase 4, protein Unc-119 homolog A, CB1 cannabinoid receptor-interacting protein 1 and serotransferrin. Expression of clusterin was found upregulated in older age samples, which was further validated by western blotting analysis. It is evident from available literature that higher expression of clusterin is associated with poor semen quality. Based on this, our study for some extent concludes and supports the fact that semen quality declines with increasing age.

Translation initiation factor IF2 contributes to ribosome assembly and maturation during cold adaptation.

Cold-stress in Escherichia coli induces de novo synthesis of translation initiation factors IF1, IF2 and IF3 while ribosome synthesis and assembly slow down. Consequently, the IFs/ribosome stoichiometric ratio increases about 3-fold during the first hours of cold adaptation. The IF1 and IF3 increase plays a role in translation regulation at low temperature (cold-shock-induced translational bias) but so far no specific role could be attributed to the extra copies of IF2. In this work, we show that the extra-copies of IF2 made after cold stress are associated with immature ribosomal subunits together with at least another nine proteins involved in assembly and/or maturation of ribosomal subunits. This finding, coupled with evidence that IF2 is endowed with GTPase-associated chaperone activity that promotes refolding of denatured GFP, and the finding that two cold-sensitive IF2 mutations cause the accumulation of immature ribosomal particles, indicate that IF2 is yet another GTPase protein that participates in ribosome assembly/maturation, especially at low temperatures. Overall, these findings are instrumental in redefining the functional role of IF2, which cannot be regarded as being restricted to its well documented functions in translation initiation of bacterial mRNA.

Transcriptional and post-transcriptional events trigger de novo infB expression in cold stressed Escherichia coli.

After a 37 to 10°C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.

Identification of Single-Nucleotide Polymorphic Loci Associated with Biomass Yield under Water Deficit in Alfalfa (Medicago sativa L.) Using Genome-Wide Sequencing and Association Mapping.

Alfalfa is a worldwide grown forage crop and is important due to its high biomass production and nutritional value. However, the production of alfalfa is challenged by adverse environmental factors such as drought and other stresses. Developing drought resistance alfalfa is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. In the present study, we used genotyping-by-sequencing and genome-wide association to identify marker loci associated with biomass yield under drought in the field in a panel of diverse germplasm of alfalfa. A total of 28 markers at 22 genetic loci were associated with yield under water deficit, whereas only four markers associated with the same trait under well-watered condition. Comparisons of marker-trait associations between water deficit and well-watered conditions showed non-similarity except one. Most of the markers were identical across harvest periods within the treatment, although different levels of significance were found among the three harvests. The loci associated with biomass yield under water deficit located throughout all chromosomes in the alfalfa genome agreed with previous reports. Our results suggest that biomass yield under drought is a complex quantitative trait with polygenic inheritance and may involve a different mechanism compared to that of non-stress. BLAST searches of the flanking sequences of the associated loci against DNA databases revealed several stress-responsive genes linked to the drought resistance loci, including leucine-rich repeat receptor-like kinase, B3 DNA-binding domain protein, translation initiation factor IF2, and phospholipase-like protein. With further investigation, those markers closely linked to drought resistance can be used for MAS to accelerate the development of new alfalfa cultivars with improved resistance to drought and other abiotic stresses.

Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin.

Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.

A model for the interaction of the G3-subdomain of Geobacillus stearothermophilus IF2 with the 30S ribosomal subunit.

Bacterial translation initiation factor IF2 complexed with GTP binds to the 30S ribosomal subunit, promotes ribosomal binding of fMet-tRNA, and favors the joining of the small and large ribosomal subunits yielding a 70S initiation complex ready to enter the translation elongation phase. Within the IF2 molecule subdomain G3, which is believed to play an important role in the IF2-30S interaction, is positioned between the GTP-binding G2 and the fMet-tRNA binding C-terminal subdomains. In this study the solution structure of subdomain G3 of Geobacillus stearothermophilus IF2 has been elucidated. G3 forms a core structure consisting of two β-sheets with each four anti-parallel strands, followed by a C-terminal α-helix. In line with its role as linker between G3 and subdomain C1, this helix has no well-defined orientation but is endowed with a dynamic nature. The structure of the G3 core is that of a typical OB-fold module, similar to that of the corresponding subdomain of Thermus thermophilus IF2, and to that of other known RNA-binding modules such as IF2-C2, IF1 and subdomains II of elongation factors EF-Tu and EF-G. Structural comparisons have resulted in a model that describes the interaction between IF2-G3 and the 30S ribosomal subunit.

Analysis of the Complete Chloroplast Genome of a Medicinal Plant, Dianthus superbus var. longicalyncinus, from a Comparative Genomics Perspective.

Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicinal plant that is also used for ornamental purposes. In this study, D. superbus was compared to its closely related family of Caryophyllaceae chloroplast (cp) genomes such as Lychnis chalcedonica and Spinacia oleracea. D. superbus had the longest large single copy (LSC) region (82,805 bp), with some variations in the inverted repeat region A (IRA)/LSC regions. The IRs underwent both expansion and constriction during evolution of the Caryophyllaceae family; however, intense variations were not identified. The pseudogene ribosomal protein subunit S19 (rps19) was identified at the IRA/LSC junction, but was not present in the cp genome of other Caryophyllaceae family members. The translation initiation factor IF-1 (infA) and ribosomal protein subunit L23 (rpl23) genes were absent from the Dianthus cp genome. When the cp genome of Dianthus was compared with 31 other angiosperm lineages, the infA gene was found to have been lost in most members of rosids, solanales of asterids and Lychnis of Caryophyllales, whereas rpl23 gene loss or pseudogization had occurred exclusively in Caryophyllales. Nevertheless, the cp genome of Dianthus and Spinacia has two introns in the proteolytic subunit of ATP-dependent protease (clpP) gene, but Lychnis has lost introns from the clpP gene. Furthermore, phylogenetic analysis of individual protein-coding genes infA and rpl23 revealed that gene loss or pseudogenization occurred independently in the cp genome of Dianthus. Molecular phylogenetic analysis also demonstrated a sister relationship between Dianthus and Lychnis based on 78 protein-coding sequences. The results presented herein will contribute to studies of the evolution, molecular biology and genetic engineering of the medicinal and ornamental plant, D. superbus var. longicalycinus.

Initiation of mRNA translation in bacteria: structural and dynamic aspects.

Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) containing initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.

Immunoproteomic profiling of Rickettsia parkeri and Rickettsia amblyommii

Rickettsia parkeri is an Amblyomma-associated, spotted fever group Rickettsia species that causes an eschar-associated, febrile illness in multiple countries throughout the Western Hemisphere. Many other rickettsial species of known or uncertain pathogenicity have been detected in Amblyomma spp. ticks in the Americas, including Rickettsia amblyommii, “Candidatus Rickettsia andeanae” and Rickettsia rickettsii. In this study, we utilized an immunoproteomic approach to compare antigenic profiles of low-passage isolates of R. parkeri and R. amblyommii with serum specimens from patients with PCR- and culture-confirmed infections with R. parkeri. Five immunoreactive proteins of R. amblyommii and nine immunoreactive proteins of R. parkeri were identified by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. Four of these, including the outer membrane protein (Omp) A, OmpB, translation initiation factor IF-2, and cell division protein FtsZ, were antigens common to both rickettsiae. Serum specimens from patients with R. parkeri rickettsiosis reacted specifically with cysteinyl–tRNA synthetase, DNA-directed RNA polymerase subunit alpha, putative sigma (54) modulation protein, chaperonin GroEL, and elongation factor Tu of R. parkeri which have been reported as virulence factors in other bacterial species. Unique antigens identified in this study may be useful for further development of the better serological assays for diagnosing infection caused by R. parkeri.

Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein.

To determine the effectiveness of evoglow-Pp1 as a reporter to study gene expression in bifidobacteria. To choose a strong and constitutive promoter to track fluorescently labelled bifidobacteria in environments under anaerobic conditions. The elongation factor P (EF-P) promoter from Bifidobacterium longum CECT 4551 produced the highest emission of fluorescence signal and was therefore able to produce the highest gene expression of the promoters studied. The promoters from B. longum CECT 4551 showed different fluorescence signal intensities which, in descending order, were: EF-P, initiation factor IF-2, elongation factor G, elongation factor Tu, elongation factor Nus A, elongation factor Ts and 30S ribosomal protein S12. The consistency of the methods employed (fluorescence imaging system, fluorescence microscopy, fluorimetry and flow cytometry) showed that the construction pNZ:Prom.GFPana contained the anaerobic fluorescent protein evoglow-Pp1 could be exploited as a tool for analysing the gene expression in bifidobacteria strains.

Ribosomal Initiation Complex-Driven Changes in the Stability and Dynamics of Initiation Factor 2 Regulate the Fidelity of Translation Initiation

Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (IC) during bacterial translation initiation is catalyzed by the initiation factor (IF) IF2. Because the rate of subunit joining is coupled to the IF, transfer RNA (tRNA), and mRNA codon compositions of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2·tRNA sub-complex forming on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF, tRNA, and codon compositions of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2·tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs undergoing rapid subunit joining display a high affinity for IF2 and an IF2·tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2·tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation.

Upregulation of eIF5B controls cell-cycle arrest and specific developmental stages.

Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met(i)) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.

Discovery of a small molecule that inhibits bacterial ribosome biogenesis.

While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15 °C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.

A Comprehensive Analysis of the Importance of Translation Initiation Factors for Haloferax volcanii Applying Deletion and Conditional Depletion Mutants

Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.

Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro

Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant T7 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg2+-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection.

Translation initiation without IF2-dependent GTP hydrolysis

Translation initiation factor IF2 is a guanine nucleotide-binding protein. The free energy change associated with guanosine triphosphate hydrolase (GTPase) activity of these proteins is believed to be the driving force allowing them to perform their functions as molecular switches. We examined role and relevance of IF2 GTPase and demonstrate that an Escherichia coli IF2 mutant bearing a single amino acid substitution (E571K) in its 30S binding domain (IF2-G3) can perform in vitro all individual translation initiation functions of wild type (wt) IF2 and supports faithful messenger RNA translation, despite having a reduced affinity for the 30S subunit and being completely inactive in GTP hydrolysis. Furthermore, the corresponding GTPase-null mutant of Bacillus stearothermophilus (E424K) can replace in vivo wt IF2 allowing an E. coli infB null mutant to grow with almost wt duplication times. Following the E571K (and E424K) mutation, which likely disrupts hydrogen bonding between subdomains G2 and G3, IF2 acquires a guanosine diphosphate (GDP)-like conformation, no longer responsive to GTP binding thereby highlighting the importance of interdomain communication in IF2. Our data underlie the importance of GTP as an IF2 ligand in the early initiation steps and the dispensability of the free energy generated by the IF2 GTPase in the late events of the translation initiation pathway.

Evolutionary and genetic analyses of mitochondrial translation initiation factors identify the missing mitochondrial IF3 in S. cerevisiae

Mitochondrial translation is essentially bacteria-like, reflecting the bacterial endosymbiotic ancestry of the eukaryotic organelle. However, unlike the translation system of its bacterial ancestors, mitochondrial translation is limited to just a few mRNAs, mainly coding for components of the respiratory complex. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in bacteria, but only IF2 is universal in mitochondria (mIF2). We analyse the distribution of mitochondrial translation initiation factors and their sequence features, given two well-propagated claims: first, a sequence insertion in mitochondrial IF2 (mIF2) compensates for the universal lack of IF1 in mitochondria, and secondly, no homologue of mitochondrial IF3 (mIF3) is identifiable in Saccharomyces cerevisiae. Our comparative sequence analysis shows that, in fact, the mIF2 insertion is highly variable and restricted in length and primary sequence conservation to vertebrates, while phylogenetic and in vivo complementation analyses reveal that an uncharacterized S. cerevisiae mitochondrial protein currently named Aim23p is a bona fide evolutionary and functional orthologue of mIF3. Our results highlight the lineage-specific nature of mitochondrial translation and emphasise that comparative analyses among diverse taxa are essential for understanding whether generalizations from model organisms can be made across eukaryotes.

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.

Structural Transitions of Translation Initiation Factor IF2 upon GDPNP and GDP Binding in Solution

Three protein factors ensure rapid and accurate initiation of translation in bacteria. Translation initiation factor IF2 is a ribosome-dependent GTPase, which is important for correct positioning of initiator tRNA on the 30S subunit as well as ribosomal subunit joining. The solution structure of the free C-terminal part of IF2 (IF2C, comprising domains IV to VI-2) was previously determined by small-angle X-ray scattering (SAXS) [Rasmussen, L. C., et al. (2008) Biochemistry 47, 5590-5598]. In this study, adding GDP or nonhydrolyzable GTP analogue GDPNP to the protein in solution caused structural changes in the protein, in agreement with recent data determined via isothermal titration calorimetry [Hauryliuk, V., et al. (2009) J. Mol. Biol. 394, 621-626]. The p(r) function indicated an elongated conformation supported by radii of gyration of 40.1 and 44.9 Å and maximum dimensions of ~125 and ~150 Å for IF2C with GDPNP and GDP, respectively. The SAXS data were used to model the structure of IF2C bound to either GDPNP or GDP. The structural transitions of IF2C upon GDPNP binding and following nucleotide hydrolysis support the concept of cofactor-dependent conformational switching rather than the classical model for GTPase activity.