The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform
Linda L. Spremulli - One of the best experts on this subject based on the ideXlab platform.
-
The interaction of mitochondrial translational Initiation Factor 2 with the small ribosomal subunit.
Biochimica et biophysica acta, 2005Co-Authors: Angela C. Spencer, Linda L. SpremulliAbstract:Bovine mitochondrial translational Initiation Factor 2 (IF-2(mt)) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III-VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2(mt) were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2(mt) was strong (K(d) approximately 10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2(mt) to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2(mt) can stabilize the binding of this Factor to 28S subunits. No binding was observed for IF-2(mt) variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2(mt). IF-2(mt) contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic Factors. Mutations in this region caused a significant reduction in the ability of the Factor to promote Initiation complex formation and to bind 28S subunits.
-
Interaction of mitochondrial Initiation Factor 2 with mitochondrial fMet-tRNA
Nucleic acids research, 2004Co-Authors: Angela C. Spencer, Linda L. SpremulliAbstract:The mammalian mitochondrial genome contains a single tRNA(Met) gene that gives rise to the initiator and elongator tRNA(Met). It is generally believed that mitochondrial protein synthesis begins with formylmethionyl-tRNA, which indicates that the formylation of mitochondrial Met-tRNA specifies its participation in Initiation through its interaction with Initiation Factor 2 (IF-2). However, recent studies in yeast mitochondria, suggest that formylation is not required for protein synthesis. In addition, bovine IF-2(mt) could replace yeast IF-2(mt) in strains that lack fMet-tRNA which suggests that this paradigm may extend to mammalian mitochondria. Here, the importance of the formylation of mitochondrial Met-tRNA for the interaction with IF-2(mt) was investigated by measuring the ability of bovine IF-2(mt) to bind mitochondrial fMet-tRNA. In direct binding experiments, bovine IF-2(mt) has a 25-fold greater affinity for mitochondrial fMet-tRNA than Met-tRNA, using either the native mitochondrial tRNA(Met) or an in vitro transcript of bovine mitochondrial tRNA(Met). In addition, IF-2(mt) will not effectively stimulate mitochondrial Met-tRNA binding to mitochondrial ribosomes, exhibiting a 50-fold preference for fMet-tRNA over Met-tRNA in this assay. Finally, the region of IF-2(mt) responsible for the interaction with fMet-tRNA was mapped to the C2 sub-domain of domain VI of this Factor.
-
Expression, purification, and mechanistic studies of bovine mitochondrial translational Initiation Factor 2.
The Journal of biological chemistry, 1996Co-Authors: Linda L. SpremulliAbstract:A complete cDNA clone encoding bovine mitochondrial translational Initiation Factor 2 (IF-2mt) has been obtained. The regions of the cDNA corresponding to mature IF-2mt and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor (approximately 90 kDa) and mature (approximately 85 kDa) forms of IF-2mt are toxic to E. coli and can only be expressed at low levels. Shorter forms of this Factor (approximately 80 and approximately 72 kDa) are also found during the expression of mature IF-2mt. The various forms of IF-2mt can be separated by high performance liquid chromatography. All of these forms are active in promoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovine mitochondrial ribosomes. IF-2mt can bind to mitochondrial ribosomes in the absence of GTP, initiator tRNA, or messenger RNA. The presence of GTP stimulates IF-2mt binding to ribosomes about 3-fold. IF-2mt interacts only weakly with GTP or with the initiator tRNA in the absence of ribosomes. Molecular dissection of IF-2mt shows that a long deletion (approximately 150 amino acid residues) from the NH2-terminal region does not affect its activity in vitro. The COOH domain of IF-2mt (amino acid residues 332-727) can bind to ribosomes even though it does not promote initiator-tRNA binding.
-
Cloning and sequence analysis of the cDNA for bovine mitochondrial translational Initiation Factor 2
Biochimica et biophysica acta, 1995Co-Authors: Mary A. Farwell, William Burkhart, Linda L. SpremulliAbstract:The complete sequence of the cDNA encoding bovine mitochondrial translational Initiation Factor 2 (IF-2mt) has been obtained by library screening followed by 3'-RACE PCR. The open reading frame for bovine IF-2mt encodes a protein of 727 amino acids. The sequence of bovine IF-2mt exhibits 85% identity to human IF-2mt, but only 38% identity to yeast IF-2mt and 39% identity to Escherichia coli IF-2 alpha.
-
Cloning and Sequence Analysis of the Human Mitochondrial Translational Initiation Factor 2 cDNA
The Journal of biological chemistry, 1995Co-Authors: Linda L. SpremulliAbstract:Complete cDNAs encoding human mitochondrial translational Initiation Factor 2 (IF-2mt) have been obtained from liver, heart, and fetal brain cDNA libraries. These cDNAs have a long open reading frame 2181 residues in length encoding a protein of 727 amino acids. Overall, human IF-2mt has 30-40% identity to the corresponding prokaryotic Factors. Surprisingly, it is no more homologous to yeast IF-2mt than to the IF-2s from bacterial sources. The greatest region of conservation lies in the G-domain of this Factor with less conservation in the COOH-terminal half of the protein and very little homology near the amino terminus. The 5'-untranslated leaders of the liver and heart cDNAs contain a number of short open reading frames. These sequences may play a role in the translational activity of the IF-2mt mRNA. Northern analysis indicates that the IF-2mt gene is expressed in all tissues but that the level of expression varies over a wide range.
Oleg Nikonov - One of the best experts on this subject based on the ideXlab platform.
-
The third structural switch in the archaeal translation Initiation Factor 2 (aIF2) molecule and its possible role in the Initiation of GTP hydrolysis and the removal of aIF2 from the ribosome.
Acta Crystallographica Section D Structural Biology, 2019Co-Authors: Oleg Nikonov, E. A. Stolboushkina, O. V. Kravchenko, Maria Garber, Natalia Nevskaya, Stanislav NikonovAbstract:The structure of the γ subunit of archaeal translation Initiation Factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ-subunit structures in the GTP-bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation Initiation Factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of Initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon-anticodon recognition.
-
Binding of the 5'-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus.
Journal of molecular biology, 2015Co-Authors: V.i. Arkhipova, E. A. Stolboushkina, Azat Gabdulkhakov, O. V. Kravchenko, Stanislav Nikonov, Maria Garber, Udo Bläsi, V. G. Kljashtorny, Birgit Märtens, Oleg NikonovAbstract:The heterotrimeric archaeal IF2 orthologue of eukaryotic translation Initiation Factor 2 consists of the α-subunit, β-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs. The biological implication of these findings is being discussed.
-
Conformational transitions in the γ subunit of the archaeal translation Initiation Factor 2
Acta Crystallographica Section D Biological Crystallography, 2014Co-Authors: Oleg Nikonov, E. A. Stolboushkina, V.i. Arkhipova, O. V. Kravchenko, Stanislav Nikonov, Maria GarberAbstract:In eukaryotes and archaea, the heterotrimeric translation Initiation Factor 2 (e/aIF2) is pivotal for the delivery of methionylated initiator tRNA (Met-tRNA(i)) to the ribosome. It acts as a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states. Recent studies show that eIF2 can also exist in a long-lived eIF2γ-GDP-P(i) (inorganic phosphate) active state. Here, four high-resolution crystal structures of aIF2γ from Sulfolobus solfataricus are reported: aIF2γ-GDPCP (a nonhydrolyzable GTP analogue), aIF2γ-GDP-formate (in which a formate ion possibly mimics P(i)), aIF2γ-GDP and nucleotide-free aIF2γ. The structures describe the different states of aIF2γ and demonstrate the conformational transitions that take place in the aIF2γ `life cycle'.
-
Crystallization of mutant forms of the γ subunit of archaeal translation Initiation Factor 2
Crystallography Reports, 2014Co-Authors: V.i. Arkhipova, E. A. Stolboushkina, Oleg Nikonov, Azat Gabdulkhakov, M. B. GarberAbstract:Archaeal translation Initiation Factor 2 (aIF2) is homologous to its eukaryotic counterpart (eIF2). It is a heterotrimeric protein consisting of α, β, and γ subunits. The protein e/aIF2 forms a ternary complex with guanosine 5′-triphosphate and the initiator methionyl-tRNA (Met-tRNAi) and delivers the latter to the ribosome. In archaea, translation Initiation Factor 2 has an additional function. The γ subunit of aIF2 binds mRNAs with a triphosphate at the 5′-end and prevents 5′-to-3′ directional mRNA decay. To determine the mRNA-binding site on the surface of aIF2γ, mutations were introduced into the protein sequence at sites of possible interactions with mRNA. The crystals of the mutant forms of aIF2γ were obtained, and X-ray diffraction data sets suitable for structure determination at atomic resolution were collected.
-
Crystal structure of the archaeal translation Initiation Factor 2 in complex with a GTP analogue and Met-tRNAf(Met.).
Journal of molecular biology, 2013Co-Authors: E. A. Stolboushkina, V.i. Arkhipova, Stanislav Nikonov, Maria Garber, Natalia Zelinskaya, Alexei Nikulin, Oleg NikonovAbstract:Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation Initiation Factor 2) in its GTP-bound form delivers Met-tRNAi(Met) to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNAi. Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ⋅GDPNP⋅Met-tRNAf(Met) has been solved at 3.2A resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation Factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-A-resolution structure of almost the same ternary Initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.
Ronald C. Wek - One of the best experts on this subject based on the ideXlab platform.
-
Eukaryotic Initiation Factor 2 Phosphorylation and Translational Control in Metabolism
Advances in nutrition (Bethesda Md.), 2012Co-Authors: Thomas D. Baird, Ronald C. WekAbstract:Regulation of mRNA translation is a rapid and effective means to couple changes in the cellular environment with global rates of protein synthesis. In response to stresses, such as nutrient deprivation and accumulation of misfolded proteins in the endoplasmic reticulum, phosphorylation of the α subunit of eukaryotic Initiation Factor 2 (eIF2α~P) reduces general translation Initiation while facilitating the preferential translation of select transcripts, such as that encoding activating transcription Factor 4 (ATF4), a transcriptional activator of genes subject to the integrated stress response (ISR). In this review, we highlight the translational control processes regulated by nutritional stress, with an emphasis on the events triggered by eIF2α~P, and describe the family of eukaryotic Initiation Factor 2 kinases and the mechanisms by which each sense different stresses. We then address 3 questions. First, what are the mechanisms by which eIF2α~P confers preferential translation on select mRNA and what are the consequences of the gene expression induced by the ISR? Second, what are the molecular processes by which certain stresses can differentially activate eIF2α~P and ATF4 expression? The third question we address is what are the modes of cross-regulation between the ISR and other stress response pathways, such as the unfolded protein response and mammalian target of rapamycin, and how do these regulatory schemes provide for gene expression programs that are tailored for specific stresses? This review highlights recent advances in each of these areas of research, emphasizing how eIF2α~P and the ISR can affect metabolic health and disease.
-
the eukaryotic Initiation Factor 2 kinase pathway facilitates differential gadd45a expression in response to environmental stress
Journal of Biological Chemistry, 2006Co-Authors: Hao Yuan Jiang, Li Jiang, Ronald C. WekAbstract:Phosphorylation of eukaryotic Initiation Factor-2 (eIF2) regulates general and gene-specific translation in response to diverse environmental stresses. Central to gene expression induced by eIF2 phosphorylation is the preferential translation of ATF4, a basic zipper transcription activator. Phosphorylation of eIF2 and its attendant induction of ATF4 can lead to different patterns of gene expression depending on the environmental stress. This is of fundamental importance because eIF2 kinases can induce the expression of genes involved in survival as well as in apoptosis. In this report, we explore the molecular basis for why there can be differential expression of GADD45a, a stress-responsive protein that regulates genome stability, apoptosis, and immune responses. We find that whereas ATF4 is required for GADD45a transcription during many different environmental stresses, GADD45a protein accumulates only during a limited number of stress arrangements. The basis for this difference between measurable GADD45a mRNA and protein lies in the observation that GADD45a protein is labile. Those stress agents that enhance ATF4-directed GADD45a transcription and impede the turnover of GADD45a protein by blocking ubiquitin/proteasome-mediated degradation elevate GADD45a protein levels. By comparison, those stress arrangements that trigger ATF4 levels and GADD45a transcription, but do not perturb the proteasome pathway, only elevate GADD45a mRNA levels. This study highlights the molecular mechanisms by which environmental stresses can differentially control central regulatory proteins targeted by the eIF2 kinase pathway.
-
parasite specific eif2 eukaryotic Initiation Factor 2 kinase required for stress induced translation control
Biochemical Journal, 2004Co-Authors: William J Sullivan, Jana Narasimhan, Micah M Bhatti, Ronald C. WekAbstract:The ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the alpha subunit of eIF2 (eukaryotic Initiation Factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2alpha (TgIF2alpha), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondii Initiation Factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2alpha in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2alpha in vitro. Finally, we demonstrate that TgIF2alpha phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.
-
ribosome binding domain of eukaryotic Initiation Factor 2 kinase gcn2 facilitates translation control
Journal of Biological Chemistry, 1998Co-Authors: Shuhao Zhu, Ronald C. WekAbstract:Abstract A family of protein kinases regulate translation Initiation in response to cellular stresses by phosphorylation of eukaryotic Initiation Factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the α subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467–1590 can bind double-stranded RNAin vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.
E. A. Stolboushkina - One of the best experts on this subject based on the ideXlab platform.
-
The third structural switch in the archaeal translation Initiation Factor 2 (aIF2) molecule and its possible role in the Initiation of GTP hydrolysis and the removal of aIF2 from the ribosome.
Acta Crystallographica Section D Structural Biology, 2019Co-Authors: Oleg Nikonov, E. A. Stolboushkina, O. V. Kravchenko, Maria Garber, Natalia Nevskaya, Stanislav NikonovAbstract:The structure of the γ subunit of archaeal translation Initiation Factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ-subunit structures in the GTP-bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation Initiation Factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of Initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon-anticodon recognition.
-
Binding of the 5'-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus.
Journal of molecular biology, 2015Co-Authors: V.i. Arkhipova, E. A. Stolboushkina, Azat Gabdulkhakov, O. V. Kravchenko, Stanislav Nikonov, Maria Garber, Udo Bläsi, V. G. Kljashtorny, Birgit Märtens, Oleg NikonovAbstract:The heterotrimeric archaeal IF2 orthologue of eukaryotic translation Initiation Factor 2 consists of the α-subunit, β-subunit and γ-subunit. Previous studies showed that the γ-subunit of aIF2, besides its central role in Met-tRNAi binding, has an additional function: it binds to the 5'-triphosphorylated end of mRNA and protects its 5'-part from degradation. Competition studies with nucleotides and mRNA, as well as structural and kinetic analyses of aIF2γ mutants, strongly implicate the canonical GTP/GDP-binding pocket in binding to the 5'-triphosphate end of mRNAs. The biological implication of these findings is being discussed.
-
Conformational transitions in the γ subunit of the archaeal translation Initiation Factor 2
Acta Crystallographica Section D Biological Crystallography, 2014Co-Authors: Oleg Nikonov, E. A. Stolboushkina, V.i. Arkhipova, O. V. Kravchenko, Stanislav Nikonov, Maria GarberAbstract:In eukaryotes and archaea, the heterotrimeric translation Initiation Factor 2 (e/aIF2) is pivotal for the delivery of methionylated initiator tRNA (Met-tRNA(i)) to the ribosome. It acts as a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states. Recent studies show that eIF2 can also exist in a long-lived eIF2γ-GDP-P(i) (inorganic phosphate) active state. Here, four high-resolution crystal structures of aIF2γ from Sulfolobus solfataricus are reported: aIF2γ-GDPCP (a nonhydrolyzable GTP analogue), aIF2γ-GDP-formate (in which a formate ion possibly mimics P(i)), aIF2γ-GDP and nucleotide-free aIF2γ. The structures describe the different states of aIF2γ and demonstrate the conformational transitions that take place in the aIF2γ `life cycle'.
-
Crystallization of mutant forms of the γ subunit of archaeal translation Initiation Factor 2
Crystallography Reports, 2014Co-Authors: V.i. Arkhipova, E. A. Stolboushkina, Oleg Nikonov, Azat Gabdulkhakov, M. B. GarberAbstract:Archaeal translation Initiation Factor 2 (aIF2) is homologous to its eukaryotic counterpart (eIF2). It is a heterotrimeric protein consisting of α, β, and γ subunits. The protein e/aIF2 forms a ternary complex with guanosine 5′-triphosphate and the initiator methionyl-tRNA (Met-tRNAi) and delivers the latter to the ribosome. In archaea, translation Initiation Factor 2 has an additional function. The γ subunit of aIF2 binds mRNAs with a triphosphate at the 5′-end and prevents 5′-to-3′ directional mRNA decay. To determine the mRNA-binding site on the surface of aIF2γ, mutations were introduced into the protein sequence at sites of possible interactions with mRNA. The crystals of the mutant forms of aIF2γ were obtained, and X-ray diffraction data sets suitable for structure determination at atomic resolution were collected.
-
Crystal structure of the archaeal translation Initiation Factor 2 in complex with a GTP analogue and Met-tRNAf(Met.).
Journal of molecular biology, 2013Co-Authors: E. A. Stolboushkina, V.i. Arkhipova, Stanislav Nikonov, Maria Garber, Natalia Zelinskaya, Alexei Nikulin, Oleg NikonovAbstract:Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation Initiation Factor 2) in its GTP-bound form delivers Met-tRNAi(Met) to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNAi. Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ⋅GDPNP⋅Met-tRNAf(Met) has been solved at 3.2A resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation Factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-A-resolution structure of almost the same ternary Initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.
Dean R. Appling - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of the C2 subdomain of yeast mitochondrial Initiation Factor 2.
Archives of biochemistry and biophysics, 2005Co-Authors: Cristiana Garofalo, Gisela Kramer, Dean R. ApplingAbstract:Abstract The COOH-terminal part of the yeast mitochondrial Initiation Factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl - Met - tRNA f Met and unformylated Met - tRNA f Met with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements. The binding of coumarin-Met-tRNA f to either protein caused a blue shift of the coumarin emission spectrum and an increase in anisotropy. Full-length ymIF2 is functionally competent in forming an Initiation complex and supporting formation of the first peptide bond on Escherichia coli ribosomes. The results demonstrate that ymIF2 has the same domain structure and biochemical properties of a typical IF2 species as found in bacteria or mammalian mitochondria—but with enhanced ability to bind unformylated initiator Met-tRNA.
-
Mammalian mitochondrial Initiation Factor 2 supports yeast mitochondrial translation without formylated initiator tRNA.
The Journal of biological chemistry, 2003Co-Authors: Anne S. Tibbetts, Lena K. Oesterlin, Sherwin Y. Chan, Gisela Kramer, Boyd Hardesty, Dean R. ApplingAbstract:Abstract Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA () in a process involving Initiation Factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial Initiation Factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated . Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.
-
Purification and characterization of yeast mitochondrial Initiation Factor 2.
Archives of biochemistry and biophysics, 2003Co-Authors: Cristiana Garofalo, Gisela Kramer, Dean R. Appling, Richard Trinko, Boyd HardestyAbstract:Yeast mitochondrial Initiation Factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this Initiation Factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.