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J W Hershey

Publications and source records attributed to J W Hershey.

At least 19 recordsLinked to original sources
PubMed2001

Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae.

Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast. Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon. To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter. After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth. Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation. A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers. The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers. Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious. Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.

Amino Acid Sequence↗
PubMed1999

A 110-kilodalton subunit of translation initiation factor eIF3 and an associated 135-kilodalton protein are encoded by the Saccharomyces cerevisiae TIF32 and TIF31 genes.

Translation initiation factor eIF3 is a multisubunit protein complex required for initiation of protein biosynthesis in eukaryotic cells. The complex promotes ribosome dissociation, the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit, and mRNA recruitment to the ribosome. In the yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in purified eIF3, we cloned the TIF31 and TIF32 genes encoding 135- (p135) and 110-kDa (p110) proteins. Deletion/disruption of TIF31 results in no change in growth rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of inhibition of the initiation phase. In addition, p110 depletion leads to p90 co-depletion, whereas other eIF3 subunit levels are not affected. Immunoprecipitation or nickel affinity chromatography from strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3 as well as p110 and p33. This establishes p110 as an authentic subunit of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses detect a direct interaction of p110 with p90, p135 with p33, and p33 with eIF4B. Our results, together with those from other laboratories, complete the cloning and characterization of all of the yeast eIF3 subunits.

Cloning, Molecular↗
PubMed1999

Characterization of the p33 subunit of eukaryotic translation initiation factor-3 from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor-3 (eIF3) is a large multisubunit complex that binds to the 40 S ribosomal subunit and promotes the binding of methionyl-tRNAi and mRNA. The molecular mechanism by which eIF3 exerts these functions is incompletely understood. We report here the cloning and characterization of TIF35, the Saccharomyces cerevisiae gene encoding the p33 subunit of eIF3. p33 is an essential protein of 30,501 Da that is required in vivo for initiation of protein synthesis. Glucose repression of TIF35 expressed from a GAL1 promoter results in depletion of both the p33 and p39 subunits. Expression of histidine-tagged p33 in yeast in combination with Ni2+ affinity chromatography allows the isolation of a complex containing the p135, p110, p90, p39, and p33 subunits of eIF3. The p33 subunit binds both mRNA and rRNA fragments due to an RNA recognition motif near its C terminus. Deletion of the C-terminal 71 amino acid residues causes loss of RNA binding, but expression of the truncated form as the sole source of p33 nevertheless supports the slow growth of yeast. These results indicate that the p33 subunit of eIF3 plays an important role in the initiation phase of protein synthesis and that its RNA-binding domain is required for optimal activity.

Amino Acid Sequence↗
PubMed1999

In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling.

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wild-type IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.

Escherichia coli↗
PubMed1999

A systematic nomenclature for new translation initiation factor genes from S. pombe and other fungi.

Eukaryotic translation initiation factors and their corresponding genes have been characterized using biochemical and genetic methods from a variety of different organisms. The designations of the factors relate to their apparent roles in the biochemical process. Many gene names indicate genetic interactions with other genes or the functional attributes used to identify them. On the other hand, progress in systematic sequencing of the genomes of organisms like Saccharomyces cerevisiae and Schizosaccharomyces pombe has revealed many genes homologous to known translation initiation factor genes. The genes defined by the systematic sequencing approach are assigned numerical designations completely unrelated to their biological function. So far there have been publications on only three genes encoding translation initiation factors from Schizosaccharomyces pombe. We therefore see this an an ideal opportunity to propose a systematic and logical nomenclature for genes encoding translation initiation factor genes that can be applied to all further genes of this type that are characterized in this fission yeast.

Fungi↗
PubMed1999

Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50-->Arg.

The molecular properties of the human eukaryotic initiation factor 5A precursor and its site directed Lys50-->Arg variant have been investigated and compared. Structure perturbation methods were used to gain information about the protein architecture in solution. Intrinsic and extrinsic spectroscopic probes strategically located in the protein matrix detected the independent unfolding of two molecular regions. Three cysteines out of four were titrated in the native protein and the peculiar presence of a tyrosinate band at neutral pH was detected. At alkaline pH only two tyrosines out of three were titratable in the native protein, with an apparent pK of about 9.9. Native protein and its Lys50-->Arg variant reacted in a similar fashion to guanidine and to pH variation, but differently to thermal stress. The complex thermal unfolding of both proteins indicated the presence of intermediates. Spectroscopic data showed that these intermediates are differently structured. Consequently, the two proteins seem to have different unfolding pathways.

Amino Acid Substitution↗
PubMed1999

Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments.

We have shown previously that p50 is the most abundant protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors. The dual function of p50 in the cell as a regulator of both transcription and translation has been suggested. To gain insight into the role of p50 in these processes, we performed the yeast two-hybrid screen to identify p50 molecular partners. Here we report the identification of actin as a p50-interacting protein. Coimmunoprecipitation of p50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between p50 and actin. Interestingly, p50 binding to actin is affected by mRNA; binding was observed at a low p50/mRNA ratio and was greatly reduced at higher ratios. Since the p50/mRNA ratio appears to be important for mRNA translatability, we speculate that p50 can regulate the attachment of mRNA to the actin network depending on its translational activity. Using immunofluorescence, we show that p50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro. Together, these findings support the view that p50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell.

3T3 Cells↗
PubMed1998

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3.

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.

Amino Acid Sequence↗
PubMed1998

Poliovirus 2A proteinase cleaves directly the eIF-4G subunit of eIF-4F complex.

The initiation of translation on eukaryotic mRNA is governed by the concerted action of polypeptides of the eIF-4F complex. One of these polypeptides, eIF-4G, is proteolytically inactivated upon infection with several members of the Picornaviridae family. This cleavage occurs by the action of virus-encoded proteinases: 2Apro (entero- and rhinovirus) or Lpro (aphthovirus). An indirect mode of eIF-4G cleavage through the activation of a second cellular proteinase has been proposed in the case of poliovirus. Although cleavage of eIF4G by rhino- and coxsackievirus 2Apro has been achieved directly in vitro, a similar activity has not been documented to date for poliovirus 2Apro. We report here that a recombinant form of poliovirus 2Apro fused to maltose binding protein (MBP) directly cleaves human eIF-4G from a highly purified eIF-4F complex. Efficient cleavage of eIF-4G requires magnesium ions. The presence of other initiation factors such as eIF-3, eIF-4A or eIF-4B mimics in part the stimulatory effect of magnesium ions and probably stabilizes the cleavage products of eIF-4G generated by 2Apro. These results suggest that efficient cleavage of eIF4G by MBP-2Apro requires a proper conformation of this factor. Finally, MBP-2Apro protein cleaves an eIF-4G-derived synthetic peptide at the same site as rhino- and coxsackievirus 2Apro (R485-G486).

Carrier Proteins↗
PubMed1998

The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro.

The major core protein of cytoplasmic messenger ribonucleoprotein particles (p50) has been shown previously to inhibit protein synthesis in vitro and in vivo. Furthermore, p50 is highly homologous to the Y-box-binding transcription factor family of proteins, binds DNA containing the Y-box motif, and thus may have a dual function in cells as a regulator of both transcription and translation. Here we show that binding or removal of p50 from rabbit reticulocyte lysate by monospecific antibodies to p50 strongly inhibits translation of endogenous and exogenous globin mRNAs as well as prokaryotic beta-galactosidase mRNA in a rabbit reticulocyte cell-free system. Thus, depending on the conditions, p50 not only may act as a translational repressor, but may also be required for protein synthesis. Translation inhibition with anti-p50 antibodies is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it does not affect elongation/termination of polypeptide chains. The inhibition with anti-p50 antibodies is followed by a decay of polysomes and accumulation of the 48 S preinitiation complex. These results suggest that p50 participates in initiation of protein biosynthesis. Although uninvolved in the formation of the 48 S preinitiation complex, p50 is necessary either for attachment of the 60 S ribosomal subunit or for previous 5'-untranslated region scanning by the 43 S preinitiation complex.

Animals↗
PubMed1997

Identification of partners of TIF34, a component of the yeast eIF3 complex, required for cell proliferation and translation initiation.

Eukaryotic initiation factor-3 (eIF3) in the yeast Saccharomyces cerevisiae plays a central role in initiation of translation. The eIF3 complex contains at least eight different proteins, but, as yet, little is known about the function of the individual proteins. In this study we have characterized the role of TIF34 (eIF3-p39), a recently identified WD-40 domain-containing protein of 39 kDa, in the eIF3 complex. Using temperature-sensitive mutants of TIF34 we show that this protein is required for cell cycle progression and for mating and plays an essential role in initiation of protein synthesis. By two-hybrid screening we have identified two partners that directly associate with TIF34: PRT1, a previously characterized eIF3 subunit, and a novel protein of 33 kDa (eIF3-p33) which is part of the eIF3 complex and has an RNA binding domain. TIF34 and p33 interact with each other and overexpression of p33 complements the growth defect of a tif34-ts mutant. Our results provide support for both physical and functional interactions between three subunits, TIF34, PRT1 and p33, in the eIF3 complex.

Amino Acid Sequence↗
PubMed1997

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Amino Acid Sequence↗
PubMed1997

The translation initiation factor eIF3-p48 subunit is encoded by int-6, a site of frequent integration by the mouse mammary tumor virus genome.

Translation initiation factor eIF3 is a large, multisubunit protein complex that plays a central role in the pathway of initiation by promoting the binding of both methionyl-tRNAi and mRNA to the 40S ribosomal subunit. As part of a broad effort to elucidate the structure of eIF3, we have cloned and sequenced the human cDNA encoding the 48-kDa subunit, eIF3-p48. The recombinant protein comigrates with the authentic p48 subunit in purified eIF3 and coprecipitates with affinity-purified antibodies to the p170 subunit of eIF3. A search of the data base indicates that the mouse gene encoding eIF3-p48 had previously been identified and characterized by others as int-6. The int-6 gene is the site of frequent integration of mouse mammary tumor virus DNA into chromosomes, implicating the gene in the regulation of cell proliferation. In addition, it was shown elsewhere that the homologous human int-6 gene product binds to the human T-cell leukemia virus type I Tax protein, leading to the translocation of Int-6 to the cytoplasm. We discuss how the cytosolic function of eIF3-p48 (Int-6) in protein synthesis may account for oncogenesis caused by these two viruses.

Amino Acid Sequence↗
PubMed1997

Overexpression in COS cells of p50, the major core protein associated with mRNA, results in translation inhibition.

p50, the major core protein of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm of somatic mammalian cells, has been characterized previously as a member of the Y-box binding transcription factor family of proteins (YB-protein) by both high structural homology and ability to bind specifically the Y-box sequence in double-stranded DNA. YB proteins are present in a whole range of cell types and some have been identified as germ-specific cytoplasmic proteins masking stored mRNA from translation. Western blot analysis of the distribution of p50 in subcellular fractions of COS-1 cells shows that p50 is a cytoplasmic protein quantitatively associated with mRNA, both in polyribosomes and in free mRNPs. The level of p50 in COS-1 cells determined by Western immunoblotting is 0.10% of total protein, which is nearly equimolar to that of ribosomes and is approximately 5-10-fold higher than the mRNA level. Transient transfection of COS-1 cells with a p50-expressing vector results in a dramatic inhibition of protein synthesis. A control transfection with a vector expressing a frameshift mutant of p50 does not cause translation inhibition. Therefore the increase in p50 protein level is responsible for the inhibitory effect in these cells.

Animals↗
PubMed1997

Conservation and diversity of eukaryotic translation initiation factor eIF3.

The largest of the mammalian translation initiation factors, eIF3, consists of at least eight subunits ranging in mass from 35 to 170 kDa. eIF3 binds to the 40 S ribosome in an early step of translation initiation and promotes the binding of methionyl-tRNAi and mRNA. We report the cloning and characterization of human cDNAs encoding two of its subunits, p110 and p36. It was found that the second slowest band during polyacrylamide gel electrophresis of eIF3 subunits in sodium dodecyl sulfate contains two proteins: p110 and p116. Analysis of the cloned cDNA encoding p110 indicates that its amino acid sequence is 31% identical to that of the yeast protein, Nip1. The p116 cDNA was cloned and characterized as a human homolog of yeast Prt1, as described elsewhere (Methot, N., Rom, E., Olsen, H., and Sonenberg, N. (1997) J. Biol. Chem. 272, 1110-1116). p36 is a WD40 repeat protein, which is 46% identical to the p39 subunit of yeast eIF3 and is identical to TRIP-1, a phosphorylation substrate of the TGF-beta type II receptor. The p116, p110, and p36 subunits localize on 40 S ribosomes in cells active in translation and co-immunoprecipitate with affinity-purified antibodies against the p170 subunit, showing that these proteins are integral components of eIF3. Although p36 and p116 have homologous protein subunits in yeast eIF3, the p110 homolog, Nip1, is not detected in yeast eIF3 preparations. The results indicate both conservation and diversity in eIF3 between yeast and humans.

Amino Acid Sequence↗
PubMed1997

The 39-kilodalton subunit of eukaryotic translation initiation factor 3 is essential for the complex's integrity and for cell viability in Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 3 (eIF3) in the yeast Saccharomyces cerevisiae comprises about eight polypeptides and plays a central role in the binding of methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The fourth largest subunit, eIF3-p39, was gel purified, and a 12-amino-acid tryptic peptide was sequenced, enabling the cloning of the TIF34 gene. TIF34 encodes a 38,753-Da protein that corresponds to eIF3-p39 in size and antigenicity. Disruption of TIF34 is lethal, and depletion of eIF3-p39 by glucose repression of TIF34 expressed from a GAL promoter results in cessation of cell growth. As eIF3-p39 levels fall, polysomes become smaller, indicating a role for eIF3-p39 in the initiation phase of protein synthesis. Unexpectedly, depletion results in degradation of all of the subunit proteins of eIF3 at a rate much faster than the normal turnover rates of these proteins. eIF3-p39 has 46% sequence identity with the p36 subunit of human eIF3. Both proteins are members of the WD-repeat family of proteins, possessing five to seven repeat elements. Taken together, the results indicate that eIF3-p39 plays an important, although not necessarily direct, role in the initiation phase of protein synthesis and suggest that it may be required for the assembly and maintenance of the eIF3 complex in eukaryotic cells.

Amino Acid Sequence↗
PubMed1996

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation in Escherichia coli.

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 in Escherichia coli, the infC, rpmI and rpIT genes encoding IF3, L35 and L20, respectively, were placed under the control of lac promotor/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express either rpmI and rplT or infC and prmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation of infC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41-43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of an rplT-lacZ gene fusion, thus confirming autogenous regulation by L20.

Bacterial Proteins↗