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H Trachsel

Publications and source records attributed to H Trachsel.

At least 37 records · Page 2Linked to original sources
PubMed1993

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.

Amino Acid Sequence↗
PubMed1992

Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.

Antibodies, Monoclonal↗
PubMed1992

Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms.

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys). Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.

Alleles↗
PubMed1992

ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.

Adenosine Triphosphate↗
PubMed1991

Expression of human recombinant lipocortin I in a wheat-germ cell-free system and Xenopus oocytes. Lipocortin is not secreted.

Lipocortin I has been presumed to be synthesized and secreted in response to glucocorticoids yet the amino acid sequence of lipocortin I reveals no signal sequence typically necessary for proteins to enter the secretory pathway. The translocation of lipocortin I across membranes was analyzed in a cell-free system and in Xenopus oocytes. Based on the published sequence, the cDNA of human lipocortin I was cloned and expressed in Escherichia coli. Lipocortin I was purified and used to raise monoclonal antibodies. To test whether lipocortin I is secreted in vitro, transcribed lipocortin mRNA was translated in a wheat germ cell-free system in the absence and presence of microsomal membranes. Prolactin mRNA was used as a control for translocation of newly synthesized protein into membrane vesicles. Prolactin, but not lipocortin I, was translocated into the membranes. To test for secretion of lipocortin I in vivo, Xenopus oocytes were co-injected with transcripts encoding lipocortin I and prolactin, with and without the signal sequence. Prolactin with the signal sequence was released into the medium. However, neither prolactin without a signal sequence nor lipocortin I was released. Carbonate extraction, using an integral transmembrane protein as control, revealed no evidence for membrane integration of lipocortin I. Thus lipocortin I is not a secreted protein.

Animals↗
PubMed1990

Translation initiation factors that function as RNA helicases from mammals, plants and yeast.

Ribosome binding to eukaryotic mRNAs requires the concerted action of three eukaryotic initiation factors: eIF-4A, eIF-4B and eIF-4F as well as the hydrolysis of ATP. These initiation factors are implicated in the unwinding of mRNA 5' secondary structure and have been isolated from mammals, yeast and wheat germ. We used an RNA unwinding assay to compare the activities of these factors from the different species. We also measured the inter-species interchangeability of these factors in the unwinding reaction. In mammals, it has been previously shown that a combination of rabbit reticulocyte eIF-4F and -4B or eIF-4A and -4B were active in the RNA unwinding assay. In wheat germ, the combination of eIF-4A and eIF-4F resulted in RNA unwinding in a reaction that was stimulated by eIF-4B. Mammalian eIF-4A was able to substitute in this system. We also show that yeast eIF-4A is able to effectively substitute for mammalian eIF-4A in duplex RNA unwinding in combination with mammalian eIF-4B, while wheat-germ eIF-4A was only partially able to substitute. Taken together, these results suggest that initiation factor requirements for RNA unwinding are largely similar in mammals, yeast and plants.

Animals↗
PubMed1990

Expression of translation initiation factor 4A from yeast and mouse in Saccharomyces cerevisiae.

The eukaryotic translation initiation factor 4A (eIF-4A) plays an important role in regulating initiation. To analyze its function in yeast, we carried out a mutational analysis of the TIF1 and TIF2 genes, which encode eIF-4A. Expression of these two yeast genes has also been investigated at the transcriptional level and it has been found that both are expressed in wild-type yeast cells. Analysis of the expression of eIF-4A-beta-galactosidase fusion proteins reveals that the TIF2 gene is more highly expressed than the TIF1 gene. Interestingly, the yeast eIF-4A protein shows a high degree of amino acid sequence similarity to the mouse homologue. However, we find that the mammalian factor does not support protein synthesis in yeast either in vivo or in vitro.

Animals↗
PubMed1990

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.

Animals↗
PubMed1990

Translation and regulation of translation in the yeast Saccharomyces cerevisiae.

In recent years the yeast Saccharomyces cerevisiae has become a model system for studies of eukaryotic translation and translation regulation. Analysis of mRNA structure, translation initiation factor sequences and the translation initiation pathway indicate, that translation in S. cerevisiae is very similar to translation in higher eukaryotes. The availability of powerful genetic techniques lead to the dissection in yeast of individual steps in the translation pathway, the detection of biochemical interactions between components involved in translation and the unravelling of complex regulation phenomena.

Gene Expression Regulation, Fungal↗
PubMed1990

The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts.

Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5'-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A. The 5'-leader sequence of TMV RNA renders translation of CAT mRNA eIF-4E-independent but still 4A-dependent.

Chloramphenicol O-Acetyltransferase↗
PubMed1989

Altered mRNA cap recognition activity of initiation factor 4E in the yeast cell cycle division mutant cdc33.

The mutation in the S. cerevisiae cell cycle division mutant cdc33 consists of a single G to A transition in the open reading frame encoding translation initiation factor 4E (eIF-4E). This leads to the substitution of glycine 113 by aspartic acid close to tryptophane 115 in the protein. This mutation reduces cap binding activity of eIF-4E as measured by binding of eIF-4E to m7GDP agarose columns and slows down overall protein synthesis at the non-permissive temperature. Comparison of the cdc33 mutation with other mutations affecting eIF-4E function supports the view that tryptophane residues and their flanking regions are involved in cap binding activity of eIF-4E.

Amino Acid Sequence↗
PubMed1989

A mammalian translation initiation factor can substitute for its yeast homologue in vivo.

The translation initiation factor 4E (eIF-4E) is involved in the recognition of the cap structure at the 5' end of eukaryotic mRNA and facilitates ribosome binding. Subsequently, additional initiation factors mediate ribosomal scanning of mRNA and initiator AUG recognition (Shatkin, A. J. (1985) Cell 40, 223-224; Rhoads, R. E. (1988) Trends Biochem. Sci. 13, 52-56; Edery, I., Pelletier, J., and Sonenberg, N. (1987) in Translational Regulation of Gene Expression (Ilan, J., ed) pp. 335-366, Plenum Publishing Corp., New York). We show here that initiation factor 4E is functionally conserved between the unicellular eukaryote Saccharomyces cerevisiae and mammals. Although the amino acid identity of the factors from both species is limited to only 33%, mouse eIF-4E can substitute for yeast eIF-4E in vivo without major effects on cell viability, growth, and mating. This finding provides a starting point for new experimental strategies to investigate the structure-function relationship of eukaryotic translation initiation factor eIF-4E.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of cap-binding proteins from yeast.

Photochemical cross-linking of Saccharomyces cerevisiae ribosomal salt wash preparations to cap-labeled mRNA reveals, in addition to the previously characterized 24-kDa cap-binding protein (eIF-4E), the presence of two novel cap-binding proteins (CBPs) of apparent molecular masses of 96 and 150 kDa. Cross-linking of the 96-kDa CBP was found to occur spontaneously without UV light induction. Based on the ATP/Mg2+ requirements, the three CBPs can be subdivided into two classes: 1) ATP/Mg2+ independent (24- and 150 kDa) and 2) Mg2+ dependent (96 kDa). The co-purification of the 24- and 150-kDa CBPs through several different chromatographic steps is consistent with the existence of a yeast CBP complex, possibly analogous to mammalian eIF-4F.

Adenine Nucleotides↗
PubMed1989

Yeast, Saccharomyces cerevisiae, cell-free translation: the inhibition of translation by high temperature is reversible.

Yeast, Saccharomyces cerevisiae, extracts are inactive for translation at 37 degrees C. Two unexplained, simultaneously occurring phenomena appear to be responsible for this effect: (i) rapid inhibition of translation, and (ii) time-dependent inactivation of (a) translational component(s) at 37 degrees C. After short incubation of an extract at 37 degrees C, protein synthesis recovers efficiently after transfer of the extract to 23 degrees C. This behaviour of yeast cell-free systems enables the in vitro inactivation of temperature-sensitive translational components and therefore facilitates studies with extracts derived from temperature-sensitive strains.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system.

Yeast Saccharomyces cerevisiae genes TIF1 and TIF2 (translation initiation factor) encode a protein tentatively called translation initiation factor (Tif) due to the similarity of its amino acid sequence and its molecular weight to mammalian eukaryotic initiation factor 4A. To clarify whether Tif is involved in translation, we produced an affinity-purified anti-Tif antibody by using Tif isolated from a Tif-overproducing yeast strain as immunogen and an Escherichia coli strain expressing Tif from an expression vector to provide the extract for affinity purification of the antibody. By using chromatographic procedures and the affinity-purified anti-Tif antibody as probe to identify Tif-containing fractions, we purified Tif from wild-type yeast cells. When yeast cells containing the only TIF1 gene on a plasmid under the control of the galactose-inducible CYC1-GAL10 promoter were grown in medium containing glucose as the carbon source, the production of Tif was shut off and growth was arrested. Lysates made from these cells were inactive in in vitro translation. Addition of Tif to these lysates restored in vitro protein synthesis. These results show that Tif is a translation factor, the yeast homologue of mammalian translation initiation factor 4A.

Blotting, Western↗
PubMed1989

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Amino Acid Sequence↗