Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

对tRNA和mRNA双依赖的兔网织红细胞无细胞蛋白合成体系的制备

本文在Gilbert和Anderson及Pel-ham和Jackson方法的基础上加以改进,制备了一个对tRNA和二RNA双依赖的兔网织红细胞无细胞蛋白合成体系。在有TMV RNA存在的情况下,加入兔网织红细胞总tRNA,氨基酸参入比不加tRNA的对照高40倍以上。在有兔网织红细胞总七RNA存在的情况下,加入TMVNA或Poly(rU),氨基酸参入分别比不加外源mRNA的对照高10倍和7倍以上。这个双依赖的无细胞蛋白合成体系是检测单一tRNA和mRNA生物活性的有用工具。     

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.     

Partial purification, characterization and translation in vitro of rat liver metallothionein messenger ribonucleic acid.

Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.     

In vitro incorporation of labelled amino acids into heart muscle ribosomes at early and late stages of compensatory heart hyperfunction]

The activity of a protein-synthesizing cell-free system from heart muscle was studied at early and late stages of compensatory heart hyperfunction. It was found that the incorporation of amino acids into heart ribosomes during 48 hours after the hyperfunction had been produced, increased by 30% as compared to the control. The incorporation of amino acids into heart ribosomes at the late stage of hyperfunction (after 6 months) was decreased by 46% as compared to the early stages. The addition of homologous tRNA to the cell-free system of protein synthesis under prolonged heart hyperfunction stimulated the incorporation of amino acids into the ribosomes by 40--50%.     

Protein-synthesizing activity of maize-shoot chromatin : I. Conditions and component requirements

The evidence presented in this paper suggests that purified plant chromatin, similar to mammalian (SR Umansky et al., Eur J Biochem 1980 105: 117-129), has the ability to incorporate amino acids into acid precipitable material. The polypeptide-synthesizing system of chromatin seems to differ substantially from the classical polyribosomal translation mechanism in cytoplasm. When chromatin purified from 5-day-old etiolated maize (Zea mays) shoots was incubated with ¹⁴C-labeled amino acids, label was incorporated into the trichloroacetic acid precipitable product. Chloramphenicol, pactamycin, and actinomycin D inhibited the incorporation almost completely, whereas treatment with cycloheximide, puromycin, or aurintricarboxylic acid did not affect the labeling. Preincubation with pancreatic RNase was also without effect, but treatment of chromatin with DNase I caused about 25% depression of label incorporation. A wheat germ translation system or its single components have no effect on the chromatin polypeptide-synthesizing activity beyond that expected for a simple addition. The protein-synthesizing system is tightly bound to chromatin and could not be removed by dissociation in 1 molar NaCl. The mean molecular weight of the major protein fraction synthesized in the presence of chromatin was 21 to 24 kilodaltons.     

Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems

Bioactive natural product peptides have diverse architectures such as non-standard sidechains and a macrocyclic backbone bearing modifications. In vitro translation of peptides bearing these features would provide the research community with a diverse collection of natural product peptide-like molecules with a potential for drug development. The ordinary in vitro translation system, however, is not amenable to the incorporation of non-proteinogenic amino acids or genetic encoding of macrocyclic backbones. To circumvent this problem, flexible tRNA-acylation ribozymes (flexizymes) were combined with a custom-made reconstituted translation system to produce the flexible in vitro translation (FIT) system. The FIT system was integrated with mRNA display to devise an in vitro selection technique, referred to as the random non-standard peptide integrated discovery (RaPID) system. It has recently yielded an N-methylated macrocyclic peptide having high affinity (Kd=0.60 nM) for its target protein, E6AP.     

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Incorporation of nonnatural amino acids into proteins by using five-base codon-anticodon pairs.

A novel strategy for the incorporation of nonnatural amino acids into proteins was developed by using five-base codon-anticodon pairs. The streptavidin mRNA containing five-base codon CGGUA and the chemically aminoacylated tRNA with five-base anticodon UACCG were prepared, and added into E. coli in vitro translation system. As a result, the nonnatural amino acid was successfully incorporated into desired position of the protein. Other five-base codons CGGN1N2, where N1 and N2 indicate one of four nucleotides, were also available for the incorporation of the nonnatural amino acid.     

Construction of modified ribosomes for incorporation of D-amino acids into proteins

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.     

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.     

Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival.

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.     

Effect of nucleoside triphosphate and magnesium ion concentration on the stability and function of rat liver polysomes in vitro

The effects of different concentrations of ATP, GTP, UTP and CTP on polysome stability and function in a cell-free protein-synthesizing system prepared from rat liver were studied. Increasing the concentration of ATP in the incubation medium to 15mm resulted in progressive disaggregation of the polysomes; at ATP concentrations above 2mm their capacity to incorporate amino acids into peptide chains diminished. The same disaggregation phenomenon could be produced by incubating polysomes in a buffered medium containing 5mm-Mg(2+) and increasing concentrations of ATP. Although the disaggregating action of ATP could be prevented by increasing Mg(2+) concentration, the amino acid incorporation in the cell-free protein-synthesizing system remained impaired. The effects of different concentrations of GTP, UTP and CTP on polysome stability were similar to those of ATP. Increasing the concentrations of each nucleoside triphosphate also inhibited the hydrolysis of GTP in the cell-free protein-synthesizing system.     

In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin-tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin-tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin-puromycin-tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast tRNA CCCG Phe. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

Expanding genetic code: Amino acids 21 and 22, selenocysteine and pyrrolysine

The discovery of two atypical amino acids, selenocysteine and pyrrolysine, in the genetic code is discussed. These findings have expanded our understanding of the genetic code, since the repertoire of amino acids in the genetic code was supplemented by two novel ones, in addition of the standard 20 amino acids. Current views on specific mechanisms of selenocysteine insertion in forming selenoproteins are considered, as well as the results of studies of new translational components involved in biosynthesis and incorporation of selenocysteine at different stages of translation. Similarity in the strategies of decoding UGA and UAG as codons for respectively selenocysteine and pyrrolysine is discussed. The review also presents evidence on the medical and biological role of selenium and selenoproteins containing selenocysteine as the main biological form of selenium.     

Interaction of translation initiation factor eIF4G with eIF4A in the yeast Saccharomyces cerevisiae.

Eukaryotic initiation factor (eIF) 4A is an essential protein that, in conjunction with eIF4B, catalyzes the ATP-dependent melting of RNA secondary structure in the 5'-untranslated region of mRNA during translation initiation. In higher eukaryotes, eIF4A is assumed to be recruited to the mRNA through its interaction with eIF4G. However, the failure to detect this interaction in yeast brought into question the generality of this model. The work presented here demonstrates that yeast eIF4G interacts with eIF4A both in vivo and in vitro. The eIF4A-binding site was mapped to amino acids 542-883 of yeast eIF4G1. Expression in yeast cells of the eIF4G1 domain that binds eIF4A results in cell growth inhibition, and addition of this domain to an eIF4A-dependent in vitro system inhibits translation in a dose-dependent manner. Both in vitro translation and cell growth can be specifically restored by increasing the eIF4A concentration. These data demonstrate that yeast eIF4A and eIF4G interact and suggest that this interaction is required for translation and cell growth.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

Flexizymes for genetic code reprogramming

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in approximately 1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.