Regulation of translation initiation and modulation of cellular physiology.

Translational control of gene expression is an important regulatory mechanism in cellular physiology. In eukaryotes, ribosomes can initiate translation by two different mechanisms: a majority of mRNAs undergo cap-dependent initiation at their extreme 5'-ends, but initiation can occur internally in some mRNAs. A number of important cellular responses, such as entry into a proliferative state and adaptation to changing nutrient levels, are mediated by changes in the mechanism of translation initiation of specific mRNAs. This article discusses new insights into control of gene expression gained through studies of regulation of eukaryotic translational initiation.

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.

mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae.

We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues. Gene disruption experiments showed that the gene is essential for growth.

A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity.

Eucaryotic initiation factor 4A (eIF-4A) is a member of a family of proteins believed to be involved in the ATP-dependent melting of RNA secondary structure. These proteins contain a derivative of the consensus ATP-binding site AXXGXGKT. To assess the importance of the consensus amino acid sequence in eIF-4A for ATP binding, we mutated the consensus amino-proximal glycine and lysine to isoleucine and asparagine, respectively. The effect of the mutations was examined by UV-induced cross-linking of [alpha-32P]dATP to eIF-4A. Mutation of the lysine residue (but not of the glycine residue) resulted in the loss of [alpha-32P]dATP cross-linking to eIF-4A, suggesting that the lysine is an important determinant in ATP binding to eIF-4A.

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.

Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family.

BACKGROUND: Translation initiation factor 4A (elF4A) is the prototype of the DEAD-box family of proteins. DEAD-box proteins are involved in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. Energy from ATP hydrolysis is used to perform RNA unwinding during initiation of mRNA translation. The presence of elF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. RESULTS: We present here the crystal structure of the nucleotide-binding domain of elF4A at 2.0 A and the structures with bound adenosinediphosphate and adenosinetriphosphate at 2.2 A and 2.4 A resolution, respectively. The structure of the apo form of the enzyme has been determined by multiple isomorphous replacement. The ATPase domain contains a central seven-stranded beta sheet flanked by nine alpha helices. Despite low sequence homology to the NTPase domains of RNA and DNA helicases, the three-dimensional fold of elF4A is nearly identical to the DNA helicase PcrA of Bacillus stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. CONCLUSIONS: We have determined the crystal structure of the N-terminal domain of the elF4A from yeast as the first structure of a member of the DEAD-box protein family. The complex of the protein with bound ADP and ATP offers insight into the mechanism of ATP hydrolysis and the transfer of energy to unwind RNA. The identical fold of the ATPase domain of the DNA helicase PcrA of B. stearothermophilus and the RNA helicase of hepatitis C virus suggests a common fold for all ATPase domains of DExx- and DEAD-box proteins.

Eukaryotic protein synthesis initiation factor eIF-3: determination of concentration and association with ribosomes in rabbit reticulocyte and HeLa cell lysates.

Monoclonal and polyclonal antibodies against eukaryotic protein synthesis initiation factor eIF-3 were produced and used to determine the factor concentration and its association with ribosomes in rabbit reticulocyte and HeLa cell lysates. In rabbit reticulocyte lysate we found 3-5 micrograms eIF-3 per mg total protein and in HeLa cell lysate 8-15 micrograms eIF-3 per mg total protein. The initiation factor eIF-3 was found both associated with 40 S ribosomal subunits and free in the post-ribosomal supernatant. However, no eIF-3 could be detected on mono- or polyribosomes.

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.

Initiation of mammalian protein synthesis. The multiple functions of the initiation factor eIF-3.

The protein synthesis initiation factor eIF-3 (a multicomponent protein complex) was labelled with 32P by phosphorylation with a protein kinase present in a partially purified 'hemin-controlled repressor' preparation. The interaction of the labelled factor with the 40 S ribosomal subunit during the course of initiation was followed. It binds to the 40 S subunit in the absence of other initiation factors and inhibits the Mg2+-dependent reassociation of the 40 S with the 60 S ribosomal subunit. It stimulates the binding of the ternary complex (eIF-2, GTP, Met-tRNAf) to the 40 S subunit, and earlier work (Trachsel, H., Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 745-767) also showed it to be essential for the subsequent binding of mRNA. The factor is released from the 40 S initiation complex during the 60 S subunit joining reaction.

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells. Stimulation by initiation factor eIF-2B.

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.

Purification of seven protein synthesis initiation factors from Krebs II ascites cells.

Seven protein synthesis initiation factors were isolated from Krebs II ascites cells using the procedures developed for the purification of the corresponding factors from rabbit reticulocytes. The ascites factors display identical characteristics in ion exchange chromatography and sucrose density gradient sedimentation. Based on their profiles in SDS polyacrylamide gels, the ascites factors have polypeptide profiles and molecular weights identical to those of the reticulocyte factors. Most significantly, each ascites factor is competent in replacing its corresponding reticulocyte factor in a reconstituted in vitro protein synthesizing system which is dependent on all seven factors.

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.

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.

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.

Electron microscopic study of eukaryotic 40S initiation complex in protein synthesis.

This electron microscopic study demonstrates that formation of a functional eukaryotic 40S initiation complex is accompanied by conformational changes which obscure the characteristic structural features of the 40S ribosomal subunits and of the initiation factor eIF-3, the only macromolecular components of the complex individually resolvable by conventional high resolution electron microscopy. The complex, characterized by a sedimentation coefficient of 46S, appears as a globular particle with a diameter of about 280 A and several characteristic protrusions and incisions. Similar structures were obtained with [40S X eIF-3] initiation complexes formed by interaction of eIF-3 from rabbit reticulocytes with 40S ribosomal subunits from either A. salina cysts or mouse liver. Incubation of eIF-3 with prokaryotic 30S subunits from E. coli produced no [30S X eIF-3] structures. The binding of eIF-3 to 40S subunits is weak, and both the [40S X eIF-3] and the complete 40S initiation complexes have to be stabilized by glutaraldehyde fixation. The extensive conformational changes associated with the complex formation preclude direct electron microscopic localization of eIF-3, a globular protein approximately 100 A in diameter, in the initiation domain of the 40S subunit.

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.

Association of an Mr 50,000 cap binding protein with the cytoskeleton in BHK cells.

A monoclonal antibody (anti-CBP antibody) is shown to be directed against cap binding protein(s) (CBP) by virtue of its ability to inhibit the translation of capped reovirus mRNA in a cell-free system derived from L-cells and inhibit the specific (cap analogue-inhibited) cross-linking of proteins to the oxidized 5' terminal cap structure of reovirus mRNA. Anti-CBP antibody reacts with an Mr 50,000 polypeptide in rabbit reticulocyte polysomes and this polypeptide appears to be associated with the 5' cap structure of mRNA. In BHK-21 cells immunofluorescence microscopy reveals that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element is further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is an Mr 50,000 component. Tryptic peptide mapping shows that this polypeptide is related to an Mr 24,000 polypeptide identified as cap binding protein in earlier experiments [Sonenberg, Morgan, Merrick & Shatkin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4843-4847].

The yeast Saccharomyces cerevisiae system: a powerful tool to study the mechanism of protein synthesis initiation in eukaryotes.

This review summarizes recent progress in the study of initiation of protein synthesis in the yeast Saccharomyces cerevisiae. Biochemical and genetic approaches provide new insight into the recognition of the 5'-end of mRNA by initiation factors and 40S ribosomes, unwinding of mRNA secondary structures in the untranslated region and proper recognition of the AUG start codon. Experiments with initiation factor-dependent cell-free systems have facilitated studies of factor functions and factor requirements for translation of different mRNAs. The analysis of mutations which suppress the inhibitory effect on translation of RNA secondary structure in the 5'-untranslated region of yeast mRNAs has led to the identification of gene products which may be involved in both transcription and translation.

Skin test and lymphocyte stimulation in delayed hypersensitivity against Staphylococcus aureus antigens. Development of hypersensitivity.

The development of delayed hypersensitivity against staphylococcal antigens in guinea pigs was observed from day 3 to day 50 after sensitization with staphylococcal homogenate in Freund's incomplete (FIA) and Freund's complete adjuvant (FCA). Skin test reactivity, stimulation of lymph node lymphocytes, and peripheral blood lymphocytes and the titre of precipitating antibodies were followed during this period. Maximal skin test reactivity as well as maximal lymphocyte responsiveness occurred at day 21 after sensitization in FCA-sensitized guinea pigs. In FIA-sensitized animals highest skin reactivity was observed at day 14 and maximal lymphocyte stimulation 35 days after sensitization. Precipitating antibodies reached a plateau at day 20 in plasma of animals sensitized with FCA and at day 35 in FIA-sensitized animals.