Comparison of dissimilarity patterns of E coli, yeast and mammalian tRNAs.

A number of experimental approaches have been developed for identification of recognition (identity) sites in tRNAs. Along with them a theoretical methodology has been proposed by McClain et al that is based on concomitant analysis of all tRNA sequences from a given species. This approach allows an evaluation of nucleotide combinations present in isoacceptor tRNAs specific for the given amino acid, and not present in equivalent positions in cloverleaf structure in other tRNAs of the same organism. These elements predicted from computer analysis of the databank could be tested experimentally for their participation in forming recognition sites. The correlation between theoretical predictions and experimental data appeared promising. The aim of the present work consisted of introducing further improvements into McClain's procedure by: i), introducing into analysis a variable region in tRNAs which had not been previously considered; to accomplish this, 'normalization' of variable nucleotides was suggested, based on primary and tertiary structures of tRNAs; ii), developing a new procedure for comparison of patterns for synonymous and non-synonymous tRNAs from different organisms; iii), analysis of 3- and 4-positional contacts between tRNAs and enzymes in addition to a formerly used 2-positional model. A systematic application of McClain's procedure to mammalian, yeast and E coli tRNAs led to the following results: i), imitancy patterns for non-synonymous tRNAs of any amino acid specificity and from any organisms analysed so far overlap by no more than 30%, providing a structural basis for discrimination with high fidelity between cognate and non-cognate tRNAs; ii), the predicted identity sites are non-randomly distributed within tRNA molecules; the dominant role is ascribed to only two regions--anticodon and amino acid stem which are located far apart from one another at extremes of all tRNA molecules; iii), the imitancy patterns for synonymous tRNAs in lower (yeast) and higher (mammalian) eukaryotes are similar but not identical; iv), distribution of predicted identity sites in the cloverleaf structure in prokaryotes and eukaryotes is essentially different: in eubacterial tRNAs the major role in recognition plays anticodon and/or amino acid acceptor stem, whereas in eukaryotic (both unicellular and multicellular) tRNAs the remaining part of the molecules is also involved in recognition; v), the imitancy patterns of synonymous tRNAs from prokaryotes and eukaryotes are dissimilar, this observation leads to the prediction that the tRNA identity sites for the same amino acid in prokaryotes and eukaryotes may differ.     

Separation of isoacceptor cysteine transfer ribonucleic acids of bakers' yeasts

Summary Isoacceptor species of certain amino acidspecific transfer ribonucleic acids (tRNAs) were fractionated by gel permeation chromatography using Sephadex G-100. The separation is attributed to the 20% ethanol-1% NaCl solvent and to the characteristics of Sephadex. Isoacceptor tRNAs specific for cysteine, arginine, phenylalanine, and histidine were recovered from commercial tRNA of yeast by this method. Highly purified cysteine-specific tRNA, obtained by a method which would not be expected to separate isoacceptor molecules when fractionated by this procedure, was shown to contain two cysteine isoacceptor tRNAs.This investigation was supported in part by Public Health Service Research Grant AM-09131 from the National Institute of Arthritis and Metabolic Diseases.     

Fluorescence-monitored conformational change on the 3'-end of tRNA upon aminoacylation.

Fluorescent tRNAs species with formycine in the 3'-terminal position (tRNA-CCF) were derived from Escherichia coli tRNA(Val). Thermus thermophilus tRNA(Aap) and Thermus thermophilus tRNA(Phe). The fluorescence of formycine was used to monitor the conformational changes at the 3'-terminus of tRNA caused by aminoacylation and hydrolysis of aminoacyl residue from aminoacyl-tRNAs. An increase of about 15% in the fluorescence intensity was observed after aminoacylation of the three tRNA-CCF. This change in fluorescence amplitude that is reversed by hydrolysis of the aminoacyl residue, does not depend on the structure of the amino acid or tRNA sequence. A local conformational change at the 3'-terminal formycine probably involving a partial destacking of the base moiety in the ACCF end takes place as a consequence of aminoacylation. A structural change at the 3'-terminus of tRNA induced by attachment and detachment of the acyl residue may be important in controlling the substrate/product relationship in reactions in which tRNA participates during protein biosynthesis.     

Effects of lymphocyte activation on transfer RNAs

The influences of mitogen activation on the functional capacity of rat splenic tRNAs were evaluated. The specific amino acid acceptor activity, pmol of a specific amino acid accepted per nmol of tRNA, of isolated splenic tRNAs from in vivo Concanavalin A (37 h)-treated rats were up to 8 times the specific amino acid acceptor activities of splenic tRNAs from control rats. Control splenic tRNAs were treated with purified liver tRNA nucleotidyltransferase in vitro to repair the 3'[CCA] terminus of tRNAs, and subsequently assayed in an aminoacylation reaction. The specific amino acid acceptor activities were slightly increased over those tRNAs not repaired with tRNA nucleotidyltransferase, indicating the presence of a low level of defective but repairable tRNAs in the control rat spleen. Furthermore, our results indicate that cyclosporin A (inhibitor of lymphocyte activation) blocks the Concanavalin A stimulation of tRNA charging ranging from 16 to 93%.     

A rapid method for determining tRNA charging levels in vivo: analysis of yeast mutants defective in the general control of amino acid biosynthesis.

We describe a simple method to quantitate the intracellular levels of charged tRNA species representing all 20 amino acids. Small RNA species are isolated from yeast cells under conditions where amino acids remain bound to their cognate tRNAs. After chromatographic removal of free amino acids, the tRNAs are discharged, and the amounts of the released amino acids are then quantitated. This method was applied to yeast cells from a wild type strain and from three mutant strains that are defective both in the general control of amino acid biosynthesis and in protein synthesis. Two of these mutant strains, previously shown to be defective in the methionine or isoleucine tRNA synthetases, respectively contain undetectable amounts of charged methionine or isoleucine although their levels of the remaining 19 amino acids are similar to a wild type strain. In contrast, a gcd1 mutant strain has normal levels of all 20 amino-acyl tRNA species. Thus, gcd1 strains are defective in general control of amino acid biosynthesis for reasons other than artifactual starvation of an amino acid due to a failure in tRNA changing.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.     

3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-32P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3'-32P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.     

Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis.

The reaction of fluorescamine with primary amino groups of tRNAs was investigated. The reagent was attached under mild conditions to the 3'-end of tRNAPhe-C-C-A(3'NH) from yeast and to the minor nucleoside x in E. coli tRNAArg, tRNALys, tRNAMet, tRNAIle and tRNAPhe. The primary aliphatic amino groups of these tRNAs react specifically so that the fluorescamine dye is not attached to the amino groups of the nucleobases. E. coli tRNA species modified on the minor nucleoside X47 can all be aminoacylated. An involvement of the minor modified nucleoside X47 in the tRNA: synthetase interaction is detected. Native tRNALys-C-C-A from E. coli can be phenylalanylated by phenylalanyl-tRNA synthetase from yeast, whereas this is not the case for fluorescamine treated tRNALys-C-C-A(XF47). Pre-tRNAPhe-C-C-A(XF47) forms a ternary complex with the elongation factor Tu:GTP from E. coli, binds enzymatically to the ribosomal A-site and is active in poly U dependent poly Phe synthesis. Fluorescamine-labelled E. coli tRNAs provide new substrates for the study of protein biosynthesis by spectroscopic methods.     

Selective 32P-labelling of individual species in a total tRNA population.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.     

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.     

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.     

Identifying the ligated amino acid of archaeal tRNAs based on positions outside the anticodon

Proper recognition of tRNAs by their aminoacyl-tRNA synthetase is essential for translation accuracy. Following evidence that the enzymes can recognize the correct tRNA even when anticodon information is masked, we search for additional nucleotide positions within the tRNA molecule that potentially contain information for amino acid identification. Analyzing 3936 sequences of tRNA genes from 86 archaeal species, we show that the tRNAs’ cognate amino acids can be identified by the information embedded in the tRNAs’ nucleotide positions without relying on the anticodon information. We present a small set of six to 10 informative positions along the tRNA, which allow for amino acid identification accuracy of 90.6% to 97.4%, respectively. We inspected tRNAs for each of the 20 amino acid types for such informative positions and found that tRNA genes for some amino acids are distinguishable from others by as few as one or two positions. The informative nucleotide positions are in agreement with nucleotide positions that were experimentally shown to affect the loaded amino acid identity. Interestingly, the knowledge gained from the tRNA genes of one archaeal phylum does not extrapolate well to another phylum. Furthermore, each species has a unique ensemble of nucleotides in the informative tRNA positions, and the similarity between the sets of positions of two distinct species reflects their evolutionary distance. Hence, we term this set of informative positions a “tRNA cipher.” It is tempting to suggest that the diverging code identified here might also serve the aminoacyl tRNA synthetase in the task of tRNA recognition.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

Aminoacylation of undermethylated mammalian transfer RNA.

To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.     

Tissue-specific differences in human transfer RNA expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.     

Transfer RNATyr of melanoma tissues and cells: relevance to melanin synthesis?

The tRNA present in swine melanoma tumor tissue and normal gray skin tissue were compared by aminoacylation of the unfractionated tRNA preparations. Of the seventeen amino acids studied, seven showed differences in rate of acceptance to tRNAs from normal and tumor tissues; the tRNAs of two amino acids, tyrosine and glycine, showed dramatic three fold increases in melanoma tumor. As melanin biosynthesis proceeds from tyrosine oxidation the investigations focused on the increase in tyrosine tRNA. Kinetic analysis of tyrosine aminoacylation to normal and melanoma tRNAs revealed no differences. Analysis of the isoaccepting species of tRNATyr from normal skin and melanoma tumor tissues identified three isoacceptors; tRNATyr, represented the predominant species in normal gray skin, while tRNA2Tyr predominated in melanoma tumor tissue. The tyrosine acceptances by tRNAs from three human melanoma cell lines were analyzed and found to be variable, but isoaccepting species analysis of the tRNATyr of these three cell lines still showed a correlation between the preponderance of tRNA2Tyr and extent of tyrosine acceptance. Additionally the enzymatic activity for the oxidation of tyrosine was found to be related to tyrosine acceptance and tRNA2Tyr predominance..     

Affinity electrophoretic detection of primary amino groups in nucleic acids: application to modified bases of tRNA and to aminoacylation.

Thiolation of primary amino groups in tRNA with the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate gives rise to species which are retarded during electrophoresis in organomercury-containing polyacrylamide gels. Since such amino groups occur, as far as is known, only as part of the modified bases 3-(3-amino-3-carboxypropyl)uridine and N-2-(5-amino-5-carboxypentyl)cytidine or as the alpha-amino group of aminoacylated tRNAs, this extension of the principle of affinity electrophoresis can be used for the detection and analysis of a specific functional group in both single tRNA species and in a mixed population. The strength of the interaction may be quantified and provides information on the chemical environment/conformation of the derivatized bases.     

Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.