Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

Overproduction and purification ofEscherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.     

The yeast cloning vector YEp13 contains a tRNA3Leu gene that can mutate to an amber suppressor

We have shown that the yeast-Escherichia coli shuttle vector YEp 13 contains, as part of its yeast chromosomal segment, a tRNA₃^Leu gene. We have also isolated and characterized a variant of YEp13, namely YEp13-a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13-a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNA₃^Leu anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNA₃^Leu gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNA₃^Leu gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53. We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNA₃^Leu genes has revealed considerable sequence homology in the immediate 5′-flanking regions of these genes.     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.     

In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNA^Leu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNA^Leu knockout strains carrying deletions of the genes for tRNA^Leu(GAG) and tRNA^Leu(UAG). Disrupting the single gene encoding tRNA^Leu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA^Leu(UAG) had a lethal effect on the yeast strain, indicating that tRNA^Leu(UAG) decoding capacity could not be compensated by another tRNA^Leu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA^Leu(UAG), a selection to identify critical tRNA^Leu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.     

Overproduction and purification of Escherichia coli tRNALeu

Chemically synthesized genes encodingEscherichia coli tRNA ₁ ^Leu and tRNA ₂ ^Leu were ligated into the plasmid pTrc99B. then transformed intoEscherichia coli MT102, respectively. The positive transformants, named MT-Leu1 and MT-Leu2, were confirmed by DNA sequencing, and the conditions of cultivation for the two transformants were optimized. As a result, leucinc accepting activity of their total tRNA reached 810 and 560 pmol/A₂₆₀, respectively: the content of tRNA ₁ ^Leu was 50% of total tRNA from MT-Leu1, while that of tRNA ₂ ^Leu was 30% of total tRNA from MT-Leu2. Both tRNA^Leus from their rotal tRNs were fractionated to 1 600 pmol/A₂₆₀ after DEAE-Sepharose and BD-cellulose column chromatography. The accurate kinetic constants of aminoacylation of the two isoacceptors of tRNA^Leu catalyzed by leucyl-tRNA synthetase were determined. Project supported by the National Natural Science Foundation of China (Grant No. 39570164).     

Primary structure of three leucine transfer RNAs from bean chloroplast

Three tRNAs^Leu have been purified from bean chloroplasts and their nucleotide sequence determined. tRNA₁^Leu has 88 nucleotides and a U¹AA anticodon, tRNA₂^Leu has 85 nucleotides and a CmAA anticodon, and tRNA₃^Leu has 83 nucleotides and a UAm⁷G anticodon.     

Structures of tobacco chloroplast genes for tRNAIle (CAU), tRNALeu (CAA), tRNACys (GCA), tRNASer (UGA) and tRNAThr (GGU): a compilation of tRNA genes from tobacco chloroplasts

Summary The location and nucleotide sequences of tobacco chloroplast genes for tRNA^Ile (CAU), tRNA^Leu (CAA), tRNA^Cys (GCA), tRNA^Ser (UGA) and tRNA^Thr (GGU) (trnI-CAU, trnL-CAA, trnC-GCA, trnS-UGA and trnT-GGU, respectively) have been determined. The trnI and trnL are located in the inverted repeat region. The trnC, trnS and trnT are present in the large single copy region. These five tRNA genes together with the 25 different tRNA genes previously published have been compiled and compared. These 30 tRNA genes corresponding to 20 amino acids are most likely to be all of the tRNA genes encoded in tobacco chloroplast genome.This paper is dedicated to Professor Morio Ikehara on the occasion of his retirement from Osaka University in March 1986.     

DNA sequences of tobacco chloroplast genes for tRNASer (GGA), tRNAThr (UGU), tRNALeu (UAA), tRNAPhe (GAA): the tRNALeu gene contains a 503 bp intron

Summary The location and nucleotide sequence of tobacco chloroplast genes for tRNA^Ser (GGA), tRNA^Thr (UGU), tRNA^Leu (UAA) and tRNA^Phe (GAA) (trnS-GGA, trnT-UGU, trnL-UAA and trnF-GAA, respectively) have been determined. These genes are located in the 10 kbp BamHI fragment which lies in the middle of the large single-copy region of the chloroplast DNA. The gene order is trnS-trnT-trnL-trnF. The trnS, trnL and trnF are encoded on the same strand while the trnT on the opposite strand. The trnL contains a 503 bp intron like maize and broad bean trnL-UAAs.     

Tritium exchange studies of transfer RNA in native and denaturated conformations

Tritium exchange was used as a probe of transfer RNA structure in experiments with unfractionated tRNA (tRNA^Unfrac and homogeneous tRNA₃^Leu from bakers' yeast. Exchange kinetics were measured over a range of ionic conditions that vary in ability to stabilize the secondary and tertiary structure of tRNA. The native conformations of both samples show the same kinetics of exchange. The kinetics for tRNA₃^Leu trapped in a denatured state in a “native” solvent are much faster, reflecting the conformation and not the ionic medium. In 0.1 M-Na⁺, where tRNA₃^Leu is denatured, the kinetics for tRNA^Unfrac are intermediate between those for native and denatured tRNA₃^Leu, suggesting that in this solvent at 0 °C some tRNAs are denatured whereas other are still native. Upon further lowering of Na⁺ concentration, tRNA^Unfrac shows increasingly faster exchange, suggesting complete electrostatic denaturation of the tertiary structure of all the tRNAs in the sample, and even disruption of secondary structure.Extrapolation of the essentially linear early-time kinetics to zero time provides minimal estimates of the number of slowly exchanging hydrogens. For native tRNA₃^Leu the number is 111±2 hydrogens, whereas for the trapped denatured conformation it is only 95±2. This difference reflects a smaller number of hydrogen-bonded bases in the denatured conformation. In 1 M-Na⁺, 101±2 slowly exchanging hydrogens are found for the native tRNA₃^Leu conformation, suggesting an incompletely formed native structure. For native tRNA^Unfrac the comparable number is 101±3. These numbers of slowly exchanging hydrogens in the native conformations are consistent with tertiary structural hydrogen-bonding. Furthermore, this tertiary structure must be responsible for the slower exchange by native tRNA. The observed numbers of exchangeable hydrogens provide a basis for comparison of hydrogen-bonding interactions in native and denatured tRNA conformations.The mechanism of renaturation was also investigated, using tritium exchange as a monitor of perturbation of base pairing during the transition. When tRNA^Unfrac in low Na⁺ is renatured by addition of Mg²⁺ during tritium exchangeout, a burst of exchange or “spillage” of tritium is detected. This suggests that a fraction of the base pairs of the rapidly renaturing tRNAs in the mixture is disrupted during renaturation. In that event, and by analogy with tRNA₃^Leu, part of the base-pairing arrangement of the denatured conformations may not be preserved in the native state; and if the native conformation includes the full “cloverleaf” pattern of secondary structure, that pattern may not be intact in some denatured conformations.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.     

Characterization of an Escherichia coli mutant, feeA, displaying resistance to the calmodulin inhibitor 48/80 and reduced expression of the rare tRNA3Leu

We previously described a mutation feeB1 conferring a temperature-sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA₃^Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30°C, and temperature-sensitive, forming short filaments at 42°C. In the feeA mutant, tRNA₃^Leu expression (but not that of tRNA₁^Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of β-galactosidase, which unusually requires the translation of 6-CUA codons, was substantially reduced, particularly at 42°C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of β-galactosidase at the non-permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA₁^Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA₃^Leu or the cellular amount of tRNA₃^Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42°C.