Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae.

Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C. Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U). Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated. The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically. Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature. Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2. These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45.

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.

Studies on the mechanism of action of a eukaryotic codon-dependent factor specific for initiator Met-tRNAf and ribosomal 40 S subunits.

A putative eukaryotic initiation factor purified from rat liver cytosol promotes the ApUpGp- and Mg2+-dependent, GTP-independent binding of initiator Met-tRNAf to ribosomal 40 S subunits. The isotopically-labeled factor binds specifically to 40 S subunits to form a binary complex which can then bind Met-tRNAf in the presence of ApUpGp. When 60 S subunits are added, an 80 S complex is formed which contains Met-tRNAf but not the factor. The Met-tRNAf bound to 80 S ribosomes reacts quantitatively with puromycin. Purified 40 S subunits exist in the form of monomers and dimers. The factor converts monomers, which appear to contain one molecule of tRNA, to a form that does not contain tRNA and dimerizes readily. Deacylated tRNA inhibits dimerization and, in the presence of the factor, converts dimers to the monomeric form; thus, the factor appears to act in a reversible manner. This protein factor may play a role in the removal of tRNA from 40 S subunits, which could be generated as a consequence of chain termination, a prerequisite to the bindig of Met-tRNAf.

The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.

Protein synthesis and aging: studies with cell-free mammalian systems.

A cell-free system devoid of polysomes, which translates natural mRNA, has been prepared from rat liver. It contains ribosomal subunits, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and protein factors necessary for translation. Protein synthesis required an energy-generating system, mRNA, and 3 mM Mg2+ concentration, and it was inhibited by 7-methylguanylic acid. The total extent and the rate of protein synthesis were approximately 30% greater when the translating system was prepared from livers of 3-month-old rats, as compared to 30-month-old rats. A ribosome-free fraction containing the protein factors required for translation was also prepared from 3-month-old and 30-month-old rat livers and brains, by extraction with 0.5 M KCl. The high-salt extracts were analyzed for elongation factors EF-1 and EF-2 in a poly(U) translating system. Although the activity of EF-2 was similar in preparations from young and old rats, the EF-1 activity in the 3-month-old rat livers and brains was 30 to 40% greater than in 30-month-old animals. The protein synthesizing activity of high salt-washed ribosomes stripped of endogenous peptidyl-tRNA and mRNA, from livers and brains of young and old animals, was the same.

Studies on native ribosomal subunits from rat liver. Purification and characterization of a ribosome dissociation factor.

A population of free, native ribosomal 40S subunits, that do not react with 60S subunits to form 80S ribosomes, has been identified in the postmicrosomal fraction of rat liver homogenates. A protein (IF-3) has been purified from high salt (0.88 M KCI) extracts of native 40S subunits by gradient centrifugation and by ammonium sulfate fractionation; it prevents the reassociation of subunits and to a limited extent dissociates ribosomes to subunits. The activity is measured by ultracentrifugation of the reaction products on linear sucrose gradients, or with an assay developed in this laboratory that couples dissociation with the 60S-specific peptidyltransferase reaction; the latter procedure measures the amount of 60S subunits released from ribosomes or remaining in incubations in the presence of IF-3. Dissociation factor activity is recovered from most of the particles that are resolved by zonal centrifugation of the total "native subunits" obtained from the postmicrosomal fraction; the highest concentration of IF-3, however, appears to be associated with native 40S subunits. The purified dissociation factor IF-3 is composed of about ten polypeptides and the molecular weight is estimated to be between 500 000 and 700 000, on the basis of glycerol and cesium chloride gradient centrifugation. When purified 40S subunits react with IF-3 or when 80S ribosomes are dissociated by IF-3, a product is formed which is dependent on the concentration of the protein factor and has the characteristics of a 40SIF-3 complex; centrifugation of the complex on sucrose and cesium chloride gradients suggests that the complex consists of 1 equiv of each of the two components. Although dissociation factor IF-3 appears to react in a specific manner with free or ribosome-associated 40S subunits, the reaction with subunits differs in several respects from that with ribosomes. The dissociation factor also appears to interact with 60S subunits but multiple complexes are formed, some with more than 1 IF-3 equiv per 60S particle. The IF-3 converts 40S dimers (55S particles) to the 40S-IF-3 complex and dissociates free, native 80S particles present in the postmicrosomal fraction, but it does not affect polysome-associated ribosomes engaged in protein synthesis.

Studies on native ribosomal subunits from rat liver. Evidence for activities associated with native 40S subunits that affect the interaction with acetylphenylalanyl-tRNA, methionyl-tRNAf, and 60S subunits.

The binding of the initiator tRNA Met-tRNAf, and of acetylphenylalanyl-tRNA, has been examined with rat liver 40S subunits derived from 80S ribosomes by dissociation with native 40S subunits sedimented from the postmicrosomal fraction and with native 40S subunits extracted with high salt-containing solutions. Binding of Met-tRNAf and acetylphenylalanyl-tRNA to derived and to salt-extracted native 40S subunits is observed in the presence of the appropriate polynucleotide template and a highly purified binding factor obtain from the soluble fraction of rat liver homogenates (R.L. IF-1). Native 40S subunits bind acetylphenylalanyl-tRNA in a reaction that requires poly(U) but not exogenous binding factor; however, Met-tRNAf is not bound to native subunits, even when supplemented with the soluble binding factor, or under conditions where factor-independent, high Mg2+-stimulated binding is observed with the derived and the salt-washed native 40S subunits. The extract obtained from native 40S subunits promotes the binding of acetylphenylalanyl-tRNA but not Met-tRNAf to derived and to salt-extracted native subunits. The addition of native 40S extract to incubations containing R.L. IF-1, Met-tRNAf, and derived 40S subunits, inhibits the formation of 40S-Met-tRNAf complex. These data suggest that the binding activity that is specific for 40S subunits and initiator tRNA, and an activity that inhibits the interaction with Met-tRNAf specifically, are both associated with native 40S subunits, and can be extracted from them by treatment with high salt-containing solutions. Derived 40S subunits react quantitatively with 60S particles to form 80S ribosomes which do not bind acetylphenylalanyl-tRNA with binding factor R.L. IF-1. Native 40S subunits react only partly with 60S subunits; about half of the native 40S subunit population forms 80S ribosomes which do not subsequently bind acetylphenylalanyl-tRNA; the remaining native 40S subunits which do not react with 60S particles bind acetylphenylalanyl-tRNA but to a lesser extent. When preformed native 40S-acetylphenylalanyl-tRNA complex is incubated with 60S subunits, about half of the subunits form an 80S-acetylphenylalanyl-tRNA complex, while the rest remains as 40S-acetylphenylalanyl-tRNA. The addition of native 40S subunit salt extract to incubations containing preformed 80S ribosomes dissociates the particles to subunits. These data suggest that in addition to the initiator tRNA binding activity and the activity that inhibits Met-tRNAf interaction, part of the native 40S subunit population also contains an activity that dissociates 80S ribosomes.