Regulation of Protein Synthesis in Zoospores of Blastocladiella

The factors responsible for the regulation of protein synthesis in the zoospores of Blastocladiella emersonii were studied by means of cell fractionation and in vitro assays. Charged transfer ribonucleic acid (tRNA) and aminoacyl-tRNA synthetases were found both inside the membrane-bound, ribosomal nuclear cap, and in the extracap cytoplasm. Ribosomes isolated from zoospore nuclear caps in low salt buffer failed to support polyuridylic acid-dependent phenylalanine incorporation. After washing with high salt buffer, the cap ribosomes were equivalent in activity to similarly prepared plant ribosomes. Both the high-salt wash from cap ribosomes and the extracap supernatant fraction contained an unidentified material which inhibited aminoacyl-tRNA binding and peptide bond formation by ribosomes. Ribosomal binding of polyuridylic acid was not inhibited. Washed cap ribosomes supported very low incorporation rates without added messenger RNA, and were highly dependent upon added poly U for phenylalanine incorporation, indicating a low level of messenger in nuclear caps. It is concluded that enclosure of the ribosomes in the nuclear cap does not in itself prevent protein synthesis, and that the lack of activity may be due to the presence of a ribosome inhibitor.     

Suppression of temperature-sensitive aminoacyl-tRNA synthetase mutations by ribosomal mutations: A possible mechanism

Summary The biochemical basis of suppression of a temperature-sensitive alanyl-tRNA synthetase (alaS) mutation by mutational alterations of the ribosome has been investigated. Measurement of the polyU-dependent polyphenylalanine synthesis showed that ribosomes from the suppressor strains are less active than ribosomes from the unsuppressed aminoacyl-tRNA synthetase mutant. In this system no increased translational ambiguity could be detected for the suppressor ribosomes. This fact and also the findings that the ram-1 mutation is not able to suppress the aminoacyl-tRNA synthetase mutation and that presence of the suppressor allele is not accompanied by a measureably improved alanyl-tRNA synthetase activity argue against the possibility that suppression might be due to increased translational misreading rates of the alanyl-tRNA synthetase mRNA.It has been further found that partial suppression of temperature sensitive growth of the alaS mutation can be achieved by independent ribosomal mutations leading to reduced growth rates because of a mutation to antibiotic resistance. Addition of low concentrations of a variety of antibiotics acting at the ribosomal level can also partially revert the temperature-sensitive phenotype of the alaS mutant. Although the possibility cannot be excluded that suppression is due to the stabilisation or activation of the mutant enzyme by some indirect effect of the suppressor ribosomal mutations, the following working hypothesis is favoured at the moment: It is assumed that limitation of the aminoacyl-tRNA synthetase activity in a certain range of the restrictive temperature causes growth inhibition by the premature termination of polypeptide synthesis at the ribosome or by the unbalanced synthesis of the individual cellular proteins under this condition. The mechanism of suppression by ribosomal mutations is proposed to consist of the release of this growth inhibition by the reduction of the rate of polypeptide synthesis, which would keep amino acid incorporation from exceeding the slow charging of tRNA and thus exhausting the pool of charged tRNA. In the suppressor strains, therefore, growth at the semi-restrictive temperature is no longer limited by the aminoacylation of tRNA but by the translational process at the mutated ribosome. This influence of the ribosomal mutation on the speed of translation could be directly or indirectly coupled with an effect on translational fidelity resulting in the prevention of the binding of uncharged or non-cognate charged tRNA or in the tighter binding of peptidyl-tRNA when cognate aminoacyl-tRNA is limiting.     

Protein synthesis and the viability of rye grains. Loss of activity of protein-synthesizing systems in vitro associated with a loss of viability

A study was made of the integrity of some components of the protein-synthesizing system from viable and non-viable embryos of rye grains. In comparison with viable-embryo components both post-ribosomal supernatant and ribosomal fractions from non-viable embryos are impaired, for neither will fully support polyphenylalanine synthesis in poly(U)-directed cell-free systems. The lesion in the supernatant lies in components other than the tRNA or the aminoacyl-tRNA synthetase, for these are as functional as those present in the fully active cell-free systems from viable embryos. The ribosomes of embryos of lowered viability show considerable fragmentation and degradation of both 18S and 25S rRNA. This breakdown does not, however, account for the complete lack of polypeptide synthesis in the poly(U)-directed non-viable-embryo system, for if provided with viable-embryo supernatant, non-viable-embryo ribosomes will sustain 60% of the viable-embryo ribosome activity. A lesion in non-viable-embryo supernatant has been located in the binding of the aminoacyl-tRNA to the ribosome. The impaired components in both supernatant and ribosomes in systems in vitro may reflect the site of faults in protein synthesis in vivo in the early hours of germination. The development of these lesions during grain storage could contribute to senescence and loss of viability in the embryos of rye.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Increase of fidelity of polypeptide synthesis by spermidine in eukaryotic cell-free systems

The mechanism of spermidine-induced increase of fidelity of polypeptide synthesis in a wheat germ cell-free system has been studied. It was found that the increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes, that reduction of misreading was more marked at the 5'-base than at the 3'-base of the codon and that misreading caused by paromomycin and kanamycin C was not significantly decreased by spermidine. It was deduced from these results that spermidine inhibited low-frequency misreading more strongly than high-frequency misreading. In addition, spermidine was found to stimulate the rejection of non-cognate aminoacyl-tRNA mainly at an initial discrimination step during the binding of amino-acyl-tRNA to ribosomes, and slightly at a subsequent GTP-dependent discrimination step, the so-called proofreading step. In yeast, rabbit reticulocyte, and Artemia salina cell-free systems, spermidine was found to increase the fidelity of protein synthesis.     

Antibacterial nitroacridine, Nitroakridin 3582: binding to nucleic acids in vitro and effects on selected cell-free model systems of macromolecular biosynthesis

Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes.     

Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation.

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.     

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.     

Some properties and the possible role of intrinsic ATPase of rat liver 80S ribosomes in peptide bond elongation

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.     

Inhibition of ribosomal translocation by peptidyl transfer ribonucleic acid analogues

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.     

Inhibition of elongation factor 1-dependent aminoacyl-tRNA binding to ribosomes by Shiga-like toxin I (VT1) from Escherichia coli O157:H7 and by Shiga toxin

A Vero toxin (VT1 or Shiga-like toxin I) from Escherichia coli O157:H7 was found to inactivate 60S ribosomal subunits of rabbit reticulocyte resulting in the inhibition of protein synthesis. The mode of inhibition of both VT1 and Shiga toxin was similar and involved the blocking of elongation-1-dependent binding of aminoacyl-tRNA of ribosomes. Non-enzymatic binding of aminoacyl-tRNA to ribosomes, peptide bond formation and translocation were not inhibited by either VT1 or Shiga toxin.     

Identification of three 30S proteins contributing to the ribosomal A site

Summary When 30S ribosomal subunits from E. coli are incubated with unfractionated 30S protein, the protein synthetic activity of the ribosomes is enhanced. Part of this effect is due to the stimulation of mRNA binding by S1 (Van Duin and Kurland, 1970). In addition, three other proteins (S2, S3 and S14) increase the number of tRNA binding sites. The enhancing effect of S2, S3 and S14 on the tRNA binding capacity of the ribosomes is seen both in the presence and absence of T factor. S2, S3 and S14 do not seem to stimulate mRNA binding. The aminoacyl-tRNA bound in response to S2, S3 and S14 is associated with the 70S ribosome and it can donate amino acid residues for polypeptide synthesis. We conclude that S2, S3 and S14 are part of the 30S A site.     

Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH(4)Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind (14)C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet.     

The effect of Cephalotaxus group alkaloids on elongation of the polypeptide chain in human ribosomes

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.     

Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal −pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme.     

The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.     

CHARACTERISTICS AND PRODUCTS OF A CELL-FREE POLYPEPTIDE SYNTHESIZING SYSTEM FROM NEONATAL AND ADULT MOUSE BRAIN

—The regulation of protein synthesis by ribosomes isolated from mouse brain tissue was studied using a cell-free polyphenylalanine synthesizing system. Polypeptide synthesis was followed by assaying translocation and analysing the reaction products by BD-cellulose chromatography. The brain ribosomal activity could be divided by these methods into two distinct steps : binding of aminoacyl-tRNA to the ribosome and active translocation leading to subsequent polyphenylalanine synthesis. In comparison to initial binding of aminoacyl-tRNA, translocation in the cell-free system increased the incorporation of labelled phenylalanine by 10-fold. An analysis of the reaction products clearly showed active ribosomal synthesis of oligophenylalanine from [³H]phe-tRNA. Ribosomes isolated from neonatal brain tissue were 2–4 times as active as those obtained from adult brain tissue in polypeptide synthesis. In addition, polypeptides synthesized on the more active ribosomes from neonates tended to be of greater chain length than those from adult. Therefore, the maturation-dependent decrease in ribosomal protein synthetic activity during neural development was shown to be directly associated with the ribosome particles.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the “accessibility” of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Reduced turnover of the elongation factor EF-1 X ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 X GTP X aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.