Exogenous tRNA Research Articles

Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

Genetic code expansion enables the incorporation of non-canonical amino acids (ncAAs) into expressed proteins. ncAAs are usually encoded by a stop codon that is decoded by an exogenous orthogonal aminoacyl tRNA synthetase and its cognate suppressor tRNA, such as the pyrrolysine {bf{text{synthetase}}}{boldsymbol{/}}{{bf{text{tRNA}}}}_{{bf{text{CUA}}}}^{{bf{text{Pyl}}}} pair. In such systems, stop codon suppression is dependent on the intracellular levels of the exogenous tRNA. Therefore, multiple copies of the tRNAPyl gene (PylT) are encoded to improve ncAA incorporation. However, certain applications in mammalian cells, such as live-cell imaging applications, where labelled tRNAs contribute to background fluorescence, can benefit from the use of less invasive minimal expression systems. Accordingly, we studied the effect of tRNAPyl on live-cell fluorescence imaging of bioorthogonally-labelled intracellular proteins. We found that in COS7 cells, a decrease in PylT copy numbers had no measurable effect on protein expression levels. Importantly, reducing PylT copy numbers improved the quality of live-cell images by enhancing the signal-to-noise ratio and reducing an immobile tRNAPyl population. This enabled us to improve live cell imaging of bioorthogonally labelled intracellular proteins, and to simultaneously label two different proteins in a cell. Our results indicate that the number of introduced PylT genes can be minimized according to the transfected cell line, incorporated ncAA, and application.

Photoinactivation of Glutamate Receptors using a Genetically Encoded Unnatural Amino Acid

Site-specific incorporation of unnatural amino acids has wide-ranging applications in the structure-function studies of proteins, offering high specificity, arbitrary environmental sensitivity of incorporation and a nearly unlimited variety of unnatural side chains. We have used photo-activatable unnatural amino acids to probe the activation and desensitization of glutamate receptors (GluRs), ligand-gated ion channels that mediate fast excitatory synaptic transmission in the brain. We introduced the unnatural amino acid cross-linker p-Benzoyl-Phenylalanine (Bpa) to the AMPA receptor GluA2 expressed in mammalian cell lines. Sites of interest were replaced by an amber stop codon (TAG). The stop codon was suppressed using a genetically-encoded exogenous tRNA and tRNA-synthetase pair that incorporate Bpa (Ye et al, 2008 J Biol Chem). We initially chose sites in the lower (D2) lobes of the LBD (S729, G725), which have been previously shown to form intermolecular disulfide cross-links that inhibit the receptor (Armstrong et al, 2006 Cell; Plested and Mayer, 2009 J. Neurosci). Patch clamp electrophysiology coupled to rapid perfusion demonstrated that Bpa-rescued GluRs had similar properties to wild-type GluRs. Receptors could be trapped in inactive conformational states upon UV irradiation and subsequent photo-cross-linking, consistent with previous work. Irreversible trapping was characterized by near-complete peak current reduction (95 %) in less than five seconds cumulative exposure to UV at 365 nm from a mercury lamp. The cross-linking rate depended on UV intensity and exposure time. Wild-type channels were unaffected by similar UV exposures. Our results demonstrate that genetically-encoded unnatural amino acids such as Bpa provide an convenient strategy to control structural rearrangements of ligand-gated ion channels in mammalian cells.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.

Preparation method forEscherichia coli S30 extracts completely dependent upon tRNA addition to catalyze cell-free protein synthesis

A simple method for depletingE. coli S30 extracts of endogenous tRNA has been developed. An ethanolamine-Sepharose® column equilibrated with water selectively captured the tRNA molecules inE. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful forin vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

TRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes.

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.

Supramolecular organization of the mammalian translation system.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.

Cell free synthesis of Toxoplasma gondii antigens

Functionally active poly(A)-containing mRNA was isolated from tachyzoites of the RH strain of Toxoplasma gondii. The T. gondii mRNA was capable of directing the synthesis of proteins in a wheat germ in vitro translation system, but not in a cell free system derived from rabbit reticulocyte lysate. Efficient translation in the wheat germ system required spermine and exogenous tRNA. Amino acid incorporation was maximal at 110 mM K + and 1.8 mM Mg 2+. Tachyzoite antigens synthesized in vitro were immunoprecipitated with T. gondii antibodies from rabbits, mice and humans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated polypeptides yielded patterns that differed according to antibody source, but all T. gondii antibody preparations reacted with a translation product with an apparent molecular weight of 24 000.

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Properties, purification, and subunit structure of class III RNA polymerases.

The class III RNA polymerases present in Xenopus laevis tissues were solubilized and partially purified by ion exchange chromatography. Single chromatographic species were detected in extracts from ovary, liver, and a kidney-derived cultured cell, whereas two chromatographic forms were detected in liver extracts. All class III enzymes analyzed exhibited characteristic responses to ionic strength changes, synthetic polynucleotide templates, and alpha-amanitin. These chromatographic and catalytic properties readily distinguished all the class III enzymes from the corresponding class I and II enzymes but clearly failed to reveal any significant differences between the various ovarian and somatic class III enzymes (with the possible exception of one of the liver enzyme forms). The ovarian RNA polymerase III was completely soluble in low ionic strength buffers and was subjected to further purification via standard procedures involving differential centrifugation, ammonium sulfate fractionation, and ion exchange chromatography. Polyacrylamide gel electrophoresis under denaturing conditions revealed at least 10 distinct polypeptides (designated subunits a-j) which were consistently present in near equimolar amounts and which ranged in size from 155,000 to 19,000 daltons. Several smaller polypeptides (less than 19,000) were also evident in all preparations but were not further characterized. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms, although subsequent analysis on denaturing gels failed to reveal any differences in polypeptide composition. From the present data, it is estimated that there are about 4 X 10(9) molecules of RNA polymerase III per oocyte nucleus. This extraordinary level of soluble RNA polymerase III in part explains the ability of oocyte nuclei (or extracts) to support the active transcription of exogenous tRNA and 5 S RNA genes. The present and previous data concerning the properties and structures of the oocyte versus somatic cell enzymes also suggest that the RNA polymerase III present in the oocyte is functionally equivalent to a somatic cell enzyme and support the previous suggestion that this enzyme is conserved and used later in (somatic) embryonic cells.

Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate

Foot-and-mouth disease (FMD) virion RNA was translated completely in a micrococcal nuclease-treated rabbit reticulocyte cell-free system. The proteins synthesized in vitro were identified (1) on polyacrylamide gels by comparison with virus-specific proteins synthesized in infected cells and authentic virion proteins and (2) on cation exchange columns by comparison of their tryptic peptides to those of virion proteins. The kinetics of appearance of the in vitro synthesized polypeptides was similar to that reported in vivo and the primary products were processed efficiently into virus-specific proteins P56, P43 (VP 0), P37, P22, and P12. Processing of primary products P50 and P93 was not as efficient in the absence of exogenous tRNA as in its presence, and only occurred after translation of a region of the genome distal to these polypeptides. The synthesis of an active proteolytic processing enzyme, apparently coded for by FMD virion RNA, was demonstrated.

Evidence for a direct role of tRNA in an amino acid transport system.

The transport of phenylalanine by the general aromatic transport system in spheroplasts of Escherichia coli 9723 has been found to be stimulated by exogenous tRNA. Neither periodate-treated tRNA nor phenylalanine-charged tRNA stimulated, and the latter inhibited, phenylalanine uptake. Among preparations of specific tRNAs, tRNAPhe and tRNATyr were effective in stimulating the uptake of phenylalanine and tyrosine, respectively, and tRNAGlu and tRNAVal gave no detectable stimulation of phenylalanine or tyrosine transport. The preparation of tRNATyr was 10 times as active as unfractionated tRNA and gave as much as 167% stimulation of tyrosine transport. Correspondingly, the preparation of tRNAPhe was at least 3.5 times as active as the unfractionated tRNA and 2.5 times as active as the preparation of tRNATyr in stimulation of phenylalanine transport. Preliminary results in fractionation of the active component of tRNA for stimulating phenylalanine uptake show that the major activity resides in minor isoacceptor(s) tRNAPhe rather than the major component tRNAPhe, and the slight activity of preparations of tRNATyr is probably due to a contamination of the active tRNAPhe. Other preliminary results indicate that this type of stimulation occurs with uptake of other amino acids and their tRNA.

Binding of tRNA to reverse transcriptase of RNA tumor viruses.

The interaction of tRNA with the reverse transcriptase (RNA-dependent DNA polymerase) of mammalian RNA viruses, such as Moloney murine leukemia virus and simian sarcoma virus, has been studied. Whereas the purified reverse transcriptase of mammalian viruses sedimented in glycerol gradients as a globular protein with a molecular weight of 70,000, after interaction with tRNA the enzyme cosedimented with a protein of 150,000 molecular weight. The twofold increase in molecular weight could be a result of either two reverse transcriptase molecules complexed with a tRNA or, alternatively, several tRNA molecules bound to a single enzyme polypeptide. The enzyme complexes were dissociated in part upon degradation of the tRNA moiety by pancreatic RNase A. The reverse transcriptase released from virions of Moloney murine leukemia virus, simian sarcoma virus, and avian myeloblastosis virus, by nonionic detergent, migrated faster on glycerol gradients than purified enzyme preparation. This phenomenon was probably due to complex formation between part of the virion enzyme and the tRNA, which is endogenous in virions. Addition of exogenous tRNA was needed, however, to quantitatively complex all the virion reverse transcriptase of Moloney murine leukemia virus and simian sarcoma viruses. The reverse transcriptase of Moloney murine leukemia virus did not show tRNA species specificity in the binding reaction when glycerol gradients were used for assay. Thus, several tRNA species of Escherichia coli, yeast, chicken, and rat origin were able to complex with the enzyme. The species specificity in the interaction between tRNA and avian myeloblastosis virus reverse transcriptase was also examined. We demonstrated that under our experimental conditions, this enzyme binds different tRNA species of E. coli and yeast as well as tRNA of chicken origin.

Influence of exogenous tRNA on growth of transplantable 32P-induced osteosarcomata

The weights of osteosarcomata that arose 22 days after the s.c. injection of cell suspensions (of whold tumours) that had been exposed in vitro to tRNA were significantly different from the weights of those arising from untreated cells. The tRNA was isolated by phenol extraction and DEAE-cellulose chromatography from eviscerated full-term rat embryos, from rat mesenchymal (granulation) tissue and from rat anaplastic osteosarcomata. Tumours developing from osteosarcoma cells treated with either embryonic or normal mesenchymal tRNA were reduced in weight by 76% and 60% respectively. These effects could not be explained as a toxic consequence, because tumour weight was increased by 128% after exposure of cells to osteosarcoma tRNA. In this osteosarcoma model it appears that tumour weights can be influenced in different ways by tRNA from different sources.

DNA-directed in vitro synthesis of β-galactosidase: Dependencies on elongation factor Tu and tRNA

The DNA-directed in vitro synthesis of β-galactosidase has been investigated in a system dependent on ribosomes, a salt wash of the ribosomes and two DEAE eluates (0.25 m and 1 m KPO 4) of the supernatant fraction. Fractionation of the 0.25 m KPO 4 DEAE eluate has made it possible to obtain elongation factor Tu (EFTu) and exogenous tRNA dependencies for β-galactosidase synthesis. The requirement for EFTu was related to the amount of tRNA present in the system. Excess amounts of tRNA caused a marked inhibition of enzyme synthesis which could, in part, be prevented when additional EFTu was added.

Methylation and aging in Rhizoctonia solani.

Our earlier studies had shown that as fungi age, many of their vital functions decrease; in Rhizoctonia solani, protein synthesis is one of the functions so affected. We now find that the ability to methylate tRNA, a vital component of the protein synthesizing system, also decreases with age. This methylation of Escherichia coli tRNA by R. solani methylase preparations increased with the concentration of enzyme and with time of incubation; in both cases the rate of increase was considerably higher for preparations from young cells than for those from old cells. The methylation reaction also increased with the concentration of substrate tRNA, with temperature, at least to 45 degrees C, and with pH to 9.0. Methylase preparations from R. solani methylated both exogenous E. coli tRNA and yeast tRNA, but were only weakly active on isolated R. solani tRNA. However, acid-precipitated methylases from R. solani were very effective in methylating the homologous exogenous tRNA. Regardless of the source of the tRNA used as substrate, the methylases from older cells were always less active than those from young cells from the same mycelium. No methylase inhibitor was detected in the fungus.

Protein Synthesis in Plant Microsomal System

AbstractAn active microsomal system from 48‐h germinating seeds of Vigna sinensis (L.) Savi has now been developed. It can incorporate amino acids into protein under both in vitro and in vivo conditions, provided dithiothreitol (a protective reagent for SH groups) and phenylthiourea (an inhibitor of phenol oxidase) are present in the buffer system for extraction; and provided the assay mixture contains added dithiothreitol.The system consists of microsomes or ribosomes, tRNA or pH 5 fraction and 20 natural amino acids, ATP and an ATP‐generating system and GTP with requirement for Mg ions. The cell fractions possess aminoacyl‐RNA synthetase activity as indicated by the aminoacylhydroxamate formation. Microsomal synthesis is stimulated by exogenous tRNA from Escheriehia coli or rat liver and sensitive to various inhibitors such as cyclo‐heximide, chloramphenicol, fusidic acid. The ribosomal transfer reaction has absolute dependence on the microsomal wash, on the crude enzyme from the same participate source, and on a synthetic messenger. It is greatly suppressed by fusidic acid and by cycloheximide.

Amino acid incorporation on 80s plant ribosomes: The complete system

A cell-free system directed by poly U or turnip yellow mosaic virus (TYMV)-RNA was obtained from imbibed seeds of Phaseolus aureus; this in vitro system was dependent upon exogenous tRNA. The poly U-directed system functioned in the presence of tRNAs from P. aureus, Vicia faba and yeast, whereas TYMV-RNA was translated only in the presence of tRNAs from P. aureus or V. faba. The pH and Mg 2+ optima for aminoacylation of tRNAs of P. aureus, V. faba and yeast by leucine and phenylalanine were related to the overall pH and ionic concentration optima for the complete system.

Aminoacyl Transfer Ribonucleic Acid Synthesis in Toluene-treated Liver Cells

SUMMARY Aminoacyl-tRNA synthetases were studied in suspensions of individual liver cells made permeable to tRNA by treat- ment with toluene. Centrifugation of the toluene-treated cells revealed that about 90% of the aminoacyl-tRNA syn- thetase activity remained associated with the cell pellet. If the toluene-treated cells were preincubated under assay conditions and then centrifuged, about ‘75% of the amino- acyl-tRNA synthetase activity remained associated with the cell pellet suggesting that aminoacylation occurred within the cell. Aminoacyl-tRNA synthesis in this system was dependent on exogenous tRNA, ATP, and Mg2+. Enzyme activity remained linear with time through about 10 min of incubation and a IO-fold increase in cell concentration. The product of the reaction was exclusively aminoacyl-tRNA. This system should prove useful for study of macromolecular synthesis in mammalian cells. Toluene treatment of bacteria has been used for a number of years to make such cells permeable to exogenous substrates (1). This procedure has received considerable attention recently in studies of DNA replication since the toluene-treated cells appear to remain relatively intact and synthesize DNA normally (2). Generally, toluene treatment has been used to render bacteria permeable to low molecular weight compounds, although it is known that pancreatic DNase can enter treated cells (2), and that the presence of Triton X-100 also makes such cells permea- ble to large proteins (3). To our knowledge, toluene treatment has not been used to study macromolecular syntheses in cells of mammalian origin. Several years ago our laboratory reported the isolation from rat liver of a high molecular weight complex containing all the aminoacyl-tRNA synthetases and a large proportion of the cellu- lar tRNA (4). In a continuation of these studies we have at- tempted to examine aminoacyl-tRNA synthetases under condi- tions which would lead to minimal disruption of the complex. For this purpose we have been working with suspensions of in- dividual liver cells made permeable by treatment with toluene. In this communication we report that aminoacyl-tRNA synthe- tases can be studied within such toluene-treated liver cells, and that these cells are permeable to exogenous tRNA. Livers were obtained from male Long-Evans rats (125 to 150 g) fed ad

Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system.

Small amounts of encephalomyocarditis virus RNA direct a 50-fold increase in amino acid incorporation, in appropriately supplemented ascites tumor cell extracts, under conditions that give rise to authentic viral polypeptides. Incorporation in these crude extracts has a novel characteristic, namely, that it is almost entirely dependent upon the addition of exogenous tRNA. Further, this incorporation is restricted in that tRNA derived from ascites tumor cells or from rat liver permits translation of viral RNA, whereas tRNA from yeast or Escherichia coli does not. These translational barriers are due, at least in part, to an incompatibility between the tRNA of yeast and E. coli and the aminoacyl-tRNA synthetases of the ascites tumor cell. A more extensive basis for this incompatibility is suggested, however, by the failure of the E. coli aminoacyl-tRNA synthetases to restore viral RNA-directed protein synthesis in the presence of tRNA from E. coli, although the coli synthetases fully restore the poly(U)-directed synthesis of polyphenyl-alanine. The possible role that unique or favored codon classes might play in this restriction is considered, together with the implications of the observed requirement for tRNA.

Uptake of exogenous tRNA by Novikoff hepatoma ascites cells.

Uptake of exogenous tRNA by Novikoff hepatoma ascites cells.