Mutant tRNA<Superscript>His</Superscript> Ineffective in Repression and Lacking Two Pseudouridine Modifications

TRANSFER RNA has been implicated in the regulation of a number of amino-acid biosynthetic operons^1–4. Histidyl-tRNA^His has been shown to be involved in regulation of the histidine operon by analysis of six genes (hisO, hisR, hisS, hisT, hisU, hisW), mutation of which causes derepression of the enzymes of the histidine biosynthetic pathway in Salmonella typhimurium^5–7. A class of derepressed mutants (hisR) has only about 55% as much tRNA^His as the wild type⁴ and in the one example sequenced, contains tRNA^HIS with a structure identical to that of the wild type⁸. Studies of mutants of the gene for histidyl-tRNA synthetase (hisS) indicated that the derepressed phenotype was associated with defects in the charging of tRNA^HISin vitro². The amounts of charged and uncharged tRNA^His present in vivo during physiological derepression of the wild type and in the six classes of regulatory mutants, have been determined⁹. This work has shown that repression of the histidine operon is correlated directly with the concentration of charged histidyl-tRNA^Hisin vivo and not with the ratio of charged to uncharged or the absolute amount of uncharged tRNA^His. The derepression observed in mutants, of hisS (the gene for histidyl-tRNA synthetase), hisR (the presumed structural gene for the single species of tRNA^His) and hisU and hisW (genes presumably involved in tRNA modification) may be explained by the lower cellular concentration of charged tRNA^His which these mutants contain.     

Primary structure of three tRNAs from brewer's yeast: tRNA2, tRNA1 and tRNA2

The primary structures of three brewer's yeast tRNAs: tRNA^Pro₂ and tRNA^His_{1 and 2} have been determined

Full-size image (14K)

The U* in the anticodon U*-G-G of tRNA^Pro₂ is probably a derivative of U; tRNA^Pro₂ has 80 per cent homology with mammalian tRNAs^Pro. tRNA^His₁ and tRNA^His₂ differ by only 5 nucleotides; they have identical anticodons and may therefore recognize both codons for histidine; they have an additional nucleotide at the 5′ end. As in all other sequenced tRNAs^His this nucleotide is not paired with the fourth nucleotide from acceptor adenosine. All three sequenced tRNAs have a low degree of homology with their counterparts from yeast mitochondria.     

The effect of the Q base modification on the usage of tRNAHis in globin synthesis

The incorporation of histidine by two competing histidine isoaccepting tRNA species into rabbit globin in a rabbit reticulocyte lysate was studied. The results show that incorporation by each isoacceptor is in proportion to its abundance, indicating that neither species is used preferentially. In a previous study (McNamara and Smith (1978) J. Biol. Chem. $\text{253}$, 5964–5970) we showed that neither tRNA^His species responds preferentially to either of the histidine codons and that there is no preferential incorporation by either species into any histidine-containing site in either globin subunit. The Q base modification is found in one tRNA^His isoacceptor while the other is hypomodified in this this characteristic. The results indicate that none of the aspects of tRNA function in translation that have been examined is affected by Q base.     

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNA^His guanylyltransferase accumulate uncharged tRNA^His lacking the G₋₁ residue and subsequently accumulate additional 5-methylcytidine (m⁵C) at residues C₄₈ and C₅₀ of tRNA^His, due to the activity of the m⁵C-methyltransferase Trm4. We show here that the increase in tRNA^His m⁵C levels does not require loss of Thg1, loss of G₋₁ of tRNA^His, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA^His m⁵C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m⁵C in tRNA^His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m⁵C on tRNA^His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m⁵C appears specific to tRNA^His, as tRNA^Val(AAC) and tRNA^Gly(GCC) have much reduced additional m⁵C during these growth arrest conditions, although they also have C₄₈ and C₅₀ and are capable of having increased m⁵C levels. Thus, tRNA^His m⁵C levels are unusually responsive to yeast growth conditions, although the significance of this additional m⁵C remains unclear.     

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.     

A deafness-associated tRNAHis mutation alters the mitochondrial function,ROS production and membrane potential

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNA^His 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNA^His. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρ^o) cells, we showed ∼70% decrease in the steady-state level of tRNA^His in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNA^His, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNA^His was observed in mutant cells. The failure in tRNA^His metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNA^His mutation leading to deafness.     

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene,and one gene for tRNAAla from Arabidopsis thaliana

Three genes and one mutant gene for tRNA^Phe (GAA) and one gene for tRNA^Ala (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNA^Phe genes are identical in their nucleotide sequence, but differ in their 5 and 3 flanking regions. The mutant tRNA^Phe (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNA^Ala gene was also determined in this experiment.     

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNA^His possess an essential extra G_–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G_–1 is added by a tRNA^His guanylyl transferase. In prokaryotes, G_–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G_–1 we find here that both maturation pathways can be used. Indeed, tRNA^His with or without a G_–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA^His precursors at both positions G₊₁ and G_–1. The G_–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA^His without G_–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA^His with a G_–1 are recovered. This shows that a previously unreported tRNA^His guanylyltransferase activity is present in plant mitochondria.     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNAf ^Met) gene was identified on a 347 bpEco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAsf ^Met or tRNAf ^Met genes studied so far. Whereas the tRNAf ^Met gene is present as a single copy in the potato mt genome, a tRNA pseudogene corresponding to 60% of a complete tRNA (from the 5 end to the variable region) and located at 105 nucleotides upstream of the tRNAf ^Met gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNAf ^Met gene in the mt genome is very different among plants, which suggests that the tRNAf ^Met gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

Human Thg1 displays tRNA-inducible GTPase activity

tRNA^His guanylyltransferase (Thg1) catalyzes the 3′-5′ incorporation of guanosine into position -1 (G-1) of tRNA^His. G-1 is unique to tRNA^His and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNA^His opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNA^His opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAs^His with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74–76), how HsThg1 prevents successive 3′-5′ incorporation of G-1/G-2/G-3 into mitochondrial tRNA^His (tRNA_m^His) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNA_m^His indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNA_m^His by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNA_m^Hisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNA_m^His.     

Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature

Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA^Lys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNA^Lys did not alter the percentages of the free-, charged- or peptidyl-tRNA^Lys species compared with those found in strains that did not overproduce tRNA^Lys. At 41°C, however, despite increases in the level of peptidyl-tRNA^Lys, the excess tRNA^Lys helped to maintain the concentration of charged-tRNA^Lys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNA^Lys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNA^Lys and the causes of tRNA^Lys starvation in pth(Ts) strains grown at non-permissive temperatures are considered.     

Mutations that overcome plasmid-mediated relaxation affect (p)ppGpp

Summary A recombinant plasmid, pMY3, was constructed in this laboratory to express the amber suppressor allele, Su⁺7, of the tRNA^Trp gene from E. coli (Yarus, 1979a). This plasmid also relaxes control of the synthesis of all stable RNA species in its host cell after amino acid deprivation. Guanosine penta and tetra-phosphate (MSII and MSI) concentrations are reduced to about one-half the levels achieved by starving the host cells carrying the cloning vehicle (pMB9) alone.We now show that the relaxation conferred on cells carrying pMY3 can be overcome by at least three different missense mutations at the chromosomal spoT locus. In these stringent, plasmid-carrying strains, the ppGpp levels attained during starvation are equivalent to or higher than that of the host cell carrying the vehicle alone.In vitro mutagenesis of the relaxing plasmid with EMS, followed by transformation and screening for plasmid-bearing stringent cells, yielded four stringent revertants of the relaxing locus. Cells carrying these mutants plasmids all have normal stringent responses to amino acid starvation, and again, elevate (p)ppGpp levels equal to or greater than 80% LS286 (pMB9) levels.Despite pMY3s modest effect on its host's MSI levels during the steady state of starvation, an obvious correlation exists between the concentration of that nucleotide and the host's ability to respond stringently. We therefore believe that the plasmid intervenes in MS metabolism. Measurements of the in vivo rates of decay of MSI and MSII after reversal of isoleucine starvation show that pMY3 has no effect on those reactions. The most likely mechanism of plasmid action is therefore inhibition of MS synthesis.Nonstandard Abbreviations MSI ppGpp - MSII pppGpp - EMS ethyl methane sulfonate - TCA trichloroacetic acid     

Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

Nucleotide sequence of the 16S-23S spacer region in the rrnA operon from a blue-green alga, Anacystis nidulans

Summary The nucleotide sequence of an entire spacer region between the 16S and 23S rRNA genes of the rrnA operon from a blue-green alga, Anacystis nidulans, has been determined. The spacer region is 545 base pairs long and encodes tRNA^fle and tRNA^Ala in the order of 16S rRNA-tRNA^fle-tRNA^Ala-23S rRNA. A striking feature is that the A. nidulans tRNA^fle gene contains no 3-CCA sequence while the tRNA^Ala gene does. These spacer tRNA genes show strong sequence homology with those of chloroplasts and bacteria.     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

The tRNATyr multigene family of Nicotiana rustica: genome organization,sequence analyses and expression in vitro

Tobacco tRNA^Tyr genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA^Tyr-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector gtWES·B and in vitro packaging. The phage library was screened with a 5-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA^Tyr. Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA^Tyr genes code for the known tobacco tRNA₁ ^Tyr (GA) and seven code for tRNA₂ ^Tyr (GA). The two tRNA species differ in one nucleotide pair at the basis of the TC stem. Only one tRNA^Tyr gene (pNtY5) contains a point mutation (T54A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs^Tyr-with one exception-are processed and spliced in both extracts. The tRNA^Tyr gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.     

3′-5′ tRNA guanylyltransferase in bacteria

The identity of the histidine specific transfer RNA (tRNA^His) is largely determined by a unique guanosine residue at position −1. In eukaryotes and archaea, the tRNA^His guanylyltransferase (Thg1) catalyzes 3′-5′ addition of G to the 5′-terminus of tRNA^His. Here, we show that Thg1 also occurs in bacteria. We demonstrate in vitro Thg1 activity for recombinant enzymes from the two bacteria Bacillus thuringiensis and Myxococcus xanthus and provide a closer investigation of several archaeal Thg1. The reaction mechanism of prokaryotic Thg1 differs from eukaryotic enzymes, as it does not require ATP. Complementation of a yeast thg1 knockout strain with bacterial Thg1 verified in vivo activity and suggests a relaxed recognition of the discriminator base in bacteria.