Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1.

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1.     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Changes in in vivo levels of charged transfer RNA species during development of the posterior silkgland of Bombyx mori

The changes in the in vivo levels of acylated tRNA specific for the amino acids that make the bulk of silf-fibroin were examined during the distinct physiological phases, the growth phase and the silk-fibroin production phase, of the developing posterior silkgland of Bombyx mori. The levels of tRNA acylated with glycine and alanine, the major amino acid components of silk-fibroin, increased about 30-fold during the transition from growth stage to fibroin production stage in each gland, whereas the increments in the levels of other aminoacylated tRNA's were substantially low. An analysis of the iso-accepting species of tRNA^Gly, the most abundant tRNA in silkgland, on benzoylated DEAE-cellulose columns showed that the levels of acylated tRNA^Gly species increased differentially during the same stages of development. These results suggested that the quantitative alterations in aminoacylated tRNA population were closely associated with the changes in protein synthesis during the terminal differentiation of silkgland.     

Identification of essential domains for Escherichia coli tRNA(leu) aminoacylation and amino acid editing using minimalist RNA molecules

Escherichia coli leucyl-tRNA synthetase (LeuRS) aminoacylates up to six different class II tRNA(leu) molecules. Each has a distinct anticodon and varied nucleotides in other regions of the tRNA. Attempts to construct a minihelix RNA that can be aminoacylated with leucine have been unsuccessful. Herein, we describe the smallest tRNA(leu) analog that has been aminoacylated to a significant extent to date. A series of tRNA(leu) analogs with various domains and combinations of domains deleted was constructed. The minimal RNA that was efficiently aminoacylated with LeuRS was one in which the anticodon stem-loop and variable arm stem-loop, but neither the D-arm nor T-arm, were deleted. Aminoacylation of this minimal RNA was abolished when the discriminator base A73 was replaced with C73 or when putative tertiary interactions between the D-loop and T-loop were disrupted, suggesting that these identity elements are still functioning in the minimized RNA. The various constructs that were significantly aminoacylated were also tested for amino acid editing by the synthetase. The anticodon and variable stem-loop domains were also dispensable for hydrolysis of the charged tRNA(leu) mimics. These results suggest that LeuRS may rely on identity elements in overlapping domains of the tRNA for both its aminoacylation and editing activities.     

Simple and rapid procedure for chromatographic identification of multiple transfer RNA species from Escherichia coli

A modification of the benzoylated DEAE-cellulose method of tRNA fractionation has been developed to provide a rapid and highly efficient method for the quantitative identification of multiple tryptophan, tyrosine, and phenylalanine tRNA species from E. coli aminoacylated in vivo. This method should be of particular use in physiological studies of these tRNA species.     

Effect of selective chemical modification of 4-thiouridine of phenylalanine transfer ribonucleic acid on enzyme recognition

Transformation of 4-thiouridine residues in Escherichia coli transfer ribonucleic acids is achieved under conditions which leave the major bases and the primary structure unaffected. The modifications of 4-thiouridine involve either alteration with N-ethylmaleimide, cyanogen bromide, or hydrogen peroxide, or a photochemical transformation effected by irradiation at 330 nm of tRNA in an organic solvent. These selective modifications were made on unfractionated species (Phe, Leu, fMet, Tyr, and Val) and purified species (Phe, fMet, and Val) of E. coli tRNA with little or no loss in their capacities to be aminoacylated. Of the tRNA species tested, subsequent treatment of 4-thiouridineless-tRNA with sodium borohydride affects only the capacity of tRNA^Phe to be aminoacylated. These observations are consistent with the proposal that the cognate ligase recognition site on tRNA^Phe is situated in the nonhydrogenbonded dihydrouridine loop area of the molecule.     

Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium

The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii. This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.     

Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

Specificity of Interaction Between the First Enzyme for Histidine Biosynthesis and Aminoacylated Histidine Transfer Ribonucleic Acid

The specificity of the interaction between phosphoribosyltransferase and partially purified preparations of various species of transfer ribonucleic acid (tRNA) was investigated with the use of a filter binding assay. The enzyme showed a higher affinity for histidyl-tRNA than for arginyl- or glutamyl-tRNA. Competition experiments revealed that the enzyme does not distinguish between the aminoacylated and deacylated forms of arginine tRNA or glutamic acid tRNA, since all the binding of the aminoacylated tRNA could be inhibited by deacylated tRNA. The enzyme does, however, distinguish between the aminoacylated and deacylated forms of histidine tRNA. Approximately 70% of the binding of aminoacylated histidine tRNA is specific, since only 30% of the binding could be inhibited by deacylated tRNA. The possibility that the regulatory role of phosphoribosyltransferase is carried out as a complex with histidyl-tRNA is consistent with these data.     

Three Different Missense Suppressor Mutations Affecting the tRNAGGGGly Species of Escherichia coli

The Escherichia coli suppressor mutation, supT, has been shown to cause a C → U substitution in the middle position of the tRNA_GGG^Gly anticodon. This is the same tRNA species that is altered by the glyUsu_AGA mutation studied previously. This finding indicates that the supT mutant tRNA reads the glutamic acid codon, GAG. The supT suppressor has also been converted to a new suppressor, called glyUsu_GAA, which will suppress the GAA mutation, trpA46. The in vivo suppression efficiencies of each of these three missense suppressors has been measured and are as follows: glyUsu_AGA, 3.6%; supT, 1.6%; and glyUsu_GAA, 0.4%. Mistranslation by these mutant glycine tRNA species has no adverse affects on cell growth since cultures possessing the suppressors grow as fast as cells without. The supT tRNA species can be observed as a peak in the profile of glycyl-tRNA fractionated on a RPC-5 chromatographic column, indicating that the mutant tRNA can be aminoacylated with reasonable efficiency. This finding contrasts with previous findings concerning the glyUsu_AGA mutant tRNA which is not significantly aminoacylated under the same conditions.     

Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage

To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

Role of methionine and formylation of initiator tRNA in initiation of protein synthesis in Escherichia coli.

We showed recently that a mutant of Escherichia coli initiator tRNA with a CAU-->CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vmax/Km(app) in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction of MetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglutamine. Consequently, a subset of mutant tRNAs which are defective in formylation and therefore inactive in initiation when they are aminoacylated with glutamine become partially active when MetRS is overproduced.     

New chromatographic and biochemical strategies for quick preparative isolation of tRNA

A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA^Phe from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA^Ile from E.coli. Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA^Ile is recovered with high specific activity. (iii) tRNA_i^Met from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.coli. The tRNA_i^Met recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.     

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.     

The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.     

Superposition of two tRNA acceptor stem crystal structures: Comparison of structure, ligands and hydration

We solved the X-ray structures of two Escherichia coli tRNA^Ser acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNA^Ser microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.