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.

Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association.

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12-IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12-IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners-all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation.

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

Sustenance of Escherichia coli on a single tRNAMet.

Living organisms possess two types of tRNAs for methionine. Initiator tRNAs bind directly into the ribosomal P-site to initiate protein synthesis, and the elongators bind to the A-site during the elongation step. Eubacterial initiators (tRNAfMet) are unique in that the methionine attached to them is formylated to facilitate their binding to initiation factor 2 (IF2), and to preclude them from binding to elongation factor Tu (EFTu). However, in mammalian mitochondria, protein synthesis proceeds with a single dual function tRNAMet. Escherichia coli possesses four tRNAfMet (initiator) and two tRNAMet (elongator) genes. Free-living organisms possessing the mitochondrion like system of single tRNAMet are unknown. We characterized mutants of E. coli tRNAfMet that function both as initiators and elongators. We show that some of the tRNAfMet mutants sustain E. coli lacking all four tRNAfMet and both tRNAMet genes, providing a basis for natural occurrence of mitochondria like situation in free living organisms. The tRNA mutants show in vivo binding to both IF2 and EFTu, indicating how they carry out these otherwise mutually exclusive functions by precise regulation of their in vivo formylation. Our results provide insights into how distinct initiator and elongator methionine tRNAs might have evolved from a single 'dual function' tRNA.

Evaluation of the infB and rpsB gene fragments as genetic markers intended for identification and phylogenetic analysis of particular representatives of the order Lactobacillales.

Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA-DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).

IF2 and unique features of initiator tRNAfMet help establish the translational reading frame.

Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNAfMet and 30S•mRNA•tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.

Emerging Helicobacter pylori levofloxacin resistance and novel genetic mutation in Nepal.

BACKGROUND: The prevalence of Helicobacter pylori antibiotic susceptibility in the Nepalese strains is untracked. We determined the antibiotic susceptibility for H. pylori and analyzed the presence of genetic mutations associated with antibiotic resistance in Nepalese strains. RESULTS: This study included 146 consecutive patients who underwent gastroduodenal endoscopy in Kathmandu, Nepal. Among 42 isolated H. pylori, there was no resistance to amoxicillin and tetracycline. In contrast, similar with typical South Asian patterns; metronidazole resistance rate in Nepalese strains were extremely high (88.1 %, 37/42). Clarithromycin resistance rate in Nepalese strains were modestly high (21.4 %, 9/42). Most of metronidazole resistant strains had highly distributed rdxA and frxA mutations, but were relative coincidence without a synergistic effect to increase the minimum inhibitory concentration (MIC). Among strains with the high MIC, 63.6 % (7/11) were associated with frameshift mutation at position 18 of frxA with or without rdxA involvement. However, based on next generation sequencing data we found that one strain with the highest MIC value had a novel mutation in the form of amino acid substituted at Ala-212, Gln-382, Ile-485 of dppA and Leu-145, Thr-168, Glu-117, Val-121, Arg-221 in dapF aside from missense mutations in full-length rdxA. Mutations at Asn-87 and/or Asp-91 of the gyrA were predominantly in levofloxacin-resistant strains. The gyrB mutation had steady relationship with the gyrA 87-91 mutations. Although three (44.4 %) and two (22.2 %) of clarithromycin resistant strains had point mutation on A2143G and A2146G, we confirmed the involvement of rpl22 and infB in high MIC strains without an 23SrRNA mutation. CONCLUSIONS: The rates of resistance to clarithromycin, metronidazole and levofloxacin were high in Nepalese strains, indicating that these antibiotics-based triple therapies are not useful as first-line treatment in Nepal. Bismuth or non-bismuth-based quadruple regimens, furazolidone-based triple therapy or rifabutin-based triple therapy may become alternative strategy in Nepal.

Stringent response processes suppress DNA damage sensitivity caused by deficiency in full-length translation initiation factor 2 or PriA helicase.

When Escherichia coli grows in the presence of DNA-damaging agents such as methyl methanesulphonate (MMS), absence of the full-length form of Translation Initiation Factor 2 (IF2-1) or deficiency in helicase activity of replication restart protein PriA leads to a considerable loss of viability. MMS sensitivity of these mutants was contingent on the stringent response alarmone (p)ppGpp being at low levels. While zero levels (ppGpp°) greatly aggravated sensitivity, high levels promoted resistance. Moreover, M+ mutations, which suppress amino acid auxotrophy of ppGpp° strains and which have been found to map to RNA polymerase subunits, largely restored resistance to IF2-1- and PriA helicase-deficient mutants. The truncated forms IF2-2/3 played a key part in inducing especially severe negative effects in ppGpp° cells when restart function priB was knocked out, causing loss of viability and severe cell filamentation, indicative of SOS induction. Even a strain with the wild-type infB allele exhibited significant filamentation and MMS sensitivity in this background whereas mutations that prevent expression of IF2-2/3 essentially eliminated filamentation and largely restored MMS resistance. The results suggest different influences of IF2-1 and IF2-2/3 on the replication restart system depending on (p)ppGpp levels, each having the capacity to maximize survival under differing growth conditions.

Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits.

We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.

A new role for translation initiation factor 2 in maintaining genome integrity.

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA-damaging agent is present.

Expression and purification of soluble human cystatin C in Escherichia coli with maltose-binding protein as a soluble partner.

Human cystatin C (CYSC) is a 13-kDa endogenous cysteine proteinase inhibitor and was investigated as a replacement for creatinine as a marker of renal function. However, expressing recombinant CYSC is difficult in Escherichia coli because of resulting low yield and insufficient purity and insolubility. Here, we cloned and fused CYSC to the C-terminus of three soluble partners - maltose-binding protein (MBP), glutathione S-transferase (GST) and translation initiation factor 2 domain I (IF2) - to screen for their ability to improve the solubility of recombinant CYSC when expressed in E. coli. MBP was best at enhancing the soluble expression of CYSC, with soluble fractions accounting for 92.8±3.11% of all proteins. For scaled production, we purified the de-tagged CYSC by using a 3C protease-cleaved MBP-T3-CYSC fused protein with immobilized metal affinity chromatography and cation-affinity purification. The molecular weights of the de-tagged CYSC and human natural CYSC were similar, and the former could react specifically with CYSC polyclonal antibody. Moreover, the de-tagged CYSC displayed full biological activity against papain and cathepsin B, which was very similar to that of the human natural CYSC protein standard. We provide a method to produce large amounts of soluble recombinant human CYSC in E. coli.

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.

Frederiksenia canicola gen. nov., sp. nov. isolated from dogs and human dog-bite wounds.

Polyphasic analysis was done on 24 strains of Bisgaard taxon 16 from five European countries and mainly isolated from dogs and human dog-bite wounds. The isolates represented a phenotypically and genetically homogenous group within the family Pasteurellaceae. Their phenotypic profile was similar to members of the genus Pasteurella. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry clearly identified taxon 16 and separated it from all other genera of Pasteurellaceae showing a characteristic peak combination. Taxon 16 can be further separated and identified by a RecN protein signature sequence detectable by a specific PCR. In all phylogenetic analyses based on 16S rRNA, rpoB, infB and recN genes, taxon 16 formed a monophyletic branch with intraspecies sequence similarity of at least 99.1, 90.8, 96.8 and 97.2 %, respectively. Taxon 16 showed closest genetic relationship with Bibersteinia trehalosi as to the 16S rRNA gene (95.9 %), the rpoB (89.8 %) and the recN (74.4 %), and with Actinobacillus lignieresii for infB (84.9 %). Predicted genome similarity values based on the recN gene sequences between taxon 16 isolates and the type strains of known genera of Pasteurellaceae were below the genus level. Major whole cell fatty acids for the strain HPA 21(T) are C14:0, C16:0, C18:0 and C16:1 ω7c/C15:0 iso 2OH. Major respiratory quinones are menaquinone-8, ubiquinone-8 and demethylmenaquinone-8. We propose to classify these organisms as a novel genus and species within the family of Pasteurellaceae named Frederiksenia canicola gen. nov., sp. nov. The type strain is HPA 21(T) (= CCUG 62410(T) = DSM 25797(T)).

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.

Assessing actual contribution of IF1, inhibitor of mitochondrial FoF1, to ATP homeostasis, cell growth, mitochondrial morphology, and cell viability.

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in mitochondria coupled with proton flow driven by the proton motive force (pmf) across membranes. It has been known that isolated IF1, an evolutionarily well conserved mitochondrial protein, can inhibit the ATP hydrolysis activity of F(o)F(1). Here, we generated HeLa cells with permanent IF1 knockdown (IF1-KD cells) and compared their energy metabolism with control cells. Under optimum growth conditions, IF1-KD cells have lower cellular ATP levels and generate a higher pmf and more reactive oxygen species. Nonetheless, IF1-KD cells and control cells show the same rates of cell growth, glucose consumption, and mitochondrial ATP synthesis. Furthermore, contrary to previous reports, the morphology of mitochondria in IF1-KD cells appears to be normal. When cells encounter sudden dissipation of pmf, the cytoplasmic ATP level in IF1-KD cells drops immediately (~1 min), whereas it remains unchanged in the control cells, indicating occurrence of futile ATP hydrolysis by F(o)F(1) in the absence of IF1. The lowered ATP level in IF1-KD cells then recovers gradually (~10 min) to the original level by consuming more glucose than control cells. The viability of IF1-KD cells and control cells is the same in the absence of pmf. Thus, IF1 contributes to ATP homeostasis, but its deficiency does not affect the growth and survival of HeLa cells. Only when cells are exposed to chemical ischemia (no glycolysis and no respiration) or high concentrations of reactive oxygen species does IF1 exhibit its ability to alleviate cell injury.

Mechanism of protein biosynthesis in mammalian mitochondria.

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Autophagy is a cell death mechanism in Toxoplasma gondii.

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust.

KEY MESSAGE: The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat. The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.

The Role of Eif2s3y in Mouse Spermatogenesis and ESC.

Eukaryotic translation initiation factor 2 subunit 3 and structural gene Y-linked (Eif2s3y) gene, the gene encoding eIF2γ protein, is located on the mouse Y chromosome short arm. The Eif2s3y gene is globally expressed in all tissues and plays an important role in regulating global and gene-specific mRNA translation initiation. During the process of protein translation initiation, Eif2s3x (its homolog) and Eif2s3y encoded eIF2γ perform similar functions. However, it has been noticed that Eif2s3y plays a crucial role in spermatogenesis, including spermatogonia mitosis, meiosis, and spermiogenesis of spermatids, which may account for infertility. In the period of spermatogenesis, the role of Eif2s3x and Eif2s3y are not equivalent. Importance of Eif2s3y has been observed in ESC and implicated in several aspects, including the pluripotency state and the proliferation rate. Here, we discuss the functional significance of Eif2s3y in mouse spermatogenesis and self-renewal of ESCs.