The Experts below are selected from a list of 42 Experts worldwide ranked by ideXlab platform
Toshio Uchiumi - One of the best experts on this subject based on the ideXlab platform.
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structural basis for translation factor recruitment to the Eukaryotic archaeal ribosomes
Journal of Biological Chemistry, 2010Co-Authors: Takao Naganuma, Naoko Nomura, Min Yao, Masahiro Mochizuki, Toshio Uchiumi, Isao TanakaAbstract:The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with Eukaryotic pentameric stalk complex; it provides access to Eukaryotic Elongation Factors at levels comparable to that of the Eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)2(P1)2(P1)2) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation Factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.
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a mode of assembly of p0 p1 and p2 proteins at the gtpase associated center in animal ribosome in vitro analyses with p0 truncation mutants
Journal of Biological Chemistry, 2005Co-Authors: Akiko Hagiya, Takao Naganuma, Yasushi Maki, Jun Ohta, Yukiko Tohkairin, Tomomi Shimizu, Takaomi Nomura, Akira Hachimori, Toshio UchiumiAbstract:Abstract Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in Eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CΔ65 and CΔ81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0·P1-P2 binding site. The mutant CΔ107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NΔ21-NΔ92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NΔ6, NΔ14, and CΔ18-CΔ81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10·L7/L12 complex and L11. Analysis of incorporation of 32P-labeled P1/P2 into the 50 S subunits with WT-P0 and CΔ81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CΔ81 bound only one copy each. The hybrid ribosome with CΔ81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on Eukaryotic Elongation Factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.
Takao Naganuma - One of the best experts on this subject based on the ideXlab platform.
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structural basis for translation factor recruitment to the Eukaryotic archaeal ribosomes
Journal of Biological Chemistry, 2010Co-Authors: Takao Naganuma, Naoko Nomura, Min Yao, Masahiro Mochizuki, Toshio Uchiumi, Isao TanakaAbstract:The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with Eukaryotic pentameric stalk complex; it provides access to Eukaryotic Elongation Factors at levels comparable to that of the Eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)2(P1)2(P1)2) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation Factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.
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a mode of assembly of p0 p1 and p2 proteins at the gtpase associated center in animal ribosome in vitro analyses with p0 truncation mutants
Journal of Biological Chemistry, 2005Co-Authors: Akiko Hagiya, Takao Naganuma, Yasushi Maki, Jun Ohta, Yukiko Tohkairin, Tomomi Shimizu, Takaomi Nomura, Akira Hachimori, Toshio UchiumiAbstract:Abstract Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in Eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CΔ65 and CΔ81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0·P1-P2 binding site. The mutant CΔ107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NΔ21-NΔ92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NΔ6, NΔ14, and CΔ18-CΔ81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10·L7/L12 complex and L11. Analysis of incorporation of 32P-labeled P1/P2 into the 50 S subunits with WT-P0 and CΔ81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CΔ81 bound only one copy each. The hybrid ribosome with CΔ81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on Eukaryotic Elongation Factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.
Ian Humpherysmith - One of the best experts on this subject based on the ideXlab platform.
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conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide mass fingerprinting
Journal of Mass Spectrometry, 1997Co-Authors: Stuart J Cordwell, Valerie C Wasinger, Anne Cerpapoljak, Mark W Duncan, Ian HumpherysmithAbstract:Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two Eukaryotic Elongation Factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.
Isao Tanaka - One of the best experts on this subject based on the ideXlab platform.
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structural basis for translation factor recruitment to the Eukaryotic archaeal ribosomes
Journal of Biological Chemistry, 2010Co-Authors: Takao Naganuma, Naoko Nomura, Min Yao, Masahiro Mochizuki, Toshio Uchiumi, Isao TanakaAbstract:The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with Eukaryotic pentameric stalk complex; it provides access to Eukaryotic Elongation Factors at levels comparable to that of the Eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)2(P1)2(P1)2) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation Factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.
Towseef Rafeequi - One of the best experts on this subject based on the ideXlab platform.
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Eukaryotic Elongation factor 2 eef2 its regulation and peptide chain Elongation
Cell Biochemistry and Function, 2011Co-Authors: Gautam Kaul, Gurulingappa Pattan, Towseef RafeequiAbstract:Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of Eukaryotic mRNAs in the cell. The Elongation stage of mRNA translation requires a substantial amount of energy and also Eukaryotic Elongation Factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up-regulated by various mechanisms in the Eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain Elongation in Eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the Elongation Factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology. Copyright © 2011 John Wiley & Sons, Ltd.