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.     

Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae.

We have isolated a mutant of Saccharomyces cerevisiae that contains 1.5% of the normal tRNA complement of isopentenyladenosine (i6A). The mutant was characterized by the reduction in efficiency of a tyrosine inserting UAA nonsense suppressor. The chromatographic profiles of tRNATyr and tRNASer on benzoylated DEAE-cellulose are consistent with the loss of i6A by these species. Transfer RNA from the mutant exhibits 6.5% of the cytokinin biological activity expected for yeast tRNA. Transfer RNAs from the mutant that normally contain i6A accept the same levels of amino acids in vitro as the fully modified species. With the exception of i6A, the level of modified bases in unfractionated tRNA from the mutant appears to be normal. The loss of i6A apparently affects tRNA's role in protein synthesis at a step subsequent to aminoacylation.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Phylogenetic comparative chemical footprint analysis of the interaction between ribonuclease P RNA and tRNA.

Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that endonucleolytically cleaves precursor sequences from the 5' ends of pre-tRNAs. The bacterial RNase P RNA-tRNA complex was examined with a footprinting approach, utilizing chemical modification to determine RNase P RNA nucleotides that potentially contact tRNA. RNase P RNA was modified with dimethylsulfate or kethoxal in the presence or absence of tRNA, and sites of modification were detected by primer extension. Comparison of the results reveals RNase P bases that are protected from modification upon binding tRNA. Analyses were carried out with RNase P RNAs from three different bacteria: Escherichia coli, Chromatium vinosum and Bacillus subtilis. Discrete bases of these RNAs that lie within conserved, homologous portions of the secondary structures are similarly protected. One protection among all three RNAs was attributed to the precursor segment of pre-tRNA. Experiments using pre-tRNAs containing precursor segments of variable length demonstrate that a precursor segment of only 2-4 nucleotides is sufficient to confer this protection. Deletion of the 3'-terminal CCA sequence of tRNA correlates with loss of protection of a particular loop in the RNase P RNA secondary structure. Analysis of mutant tRNAs containing sequential 3'-terminal deletions suggests a relative orientation of the bound tRNA CCA to that loop.     

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.     

The recognition by RNase P of precursor tRNAs

We have generated mutants of M1 RNA, the catalytic subunit of Escherichia coli RNaseP, and have analyzed their properties in vitro and in vivo. The mutations, A333----C333, A334----U334, and A333 A334----C333 U334 are within the sequence UGAAU which is complementary to the GT psi CR sequence found in loop IV of all E. coli tRNAs. We have examined: 1) whether the mutant M1 RNAs are active in processing wild type tRNA precursors and 2) whether they can restore the processing defect in mutant tRNA precursors with changes within the GT psi CR sequence. As substrates for in vitro studies we used wild type E. coli SuIII tRNA(Tyr) precursor, and pTyrA54, a mutant tRNA precursor with a base change that could potentially complement the U334 mutation in M1 RNA. The C333 mutation had no effect on activity of M1 RNA on wild type pTyr. The U334 mutant M1 RNA, on the other hand, had a much lower activity on wild type pTyr. However, use of pTyrA54 as substrate instead of wild type pTyr did not restore the activity of the U334 mutant M1 RNA. These results suggest that interactions via base pairing between nucleotides 331-335 of M1 RNA and the GT psi CG of pTyr are probably not essential for cleavage of these tRNA precursors by M1 RNA. For assays of in vivo function, we examined the ability of mutant M1 RNAs to complement a ts mutation in the protein component of RNaseP in FS101, a recA- derivative of E. coli strain A49. In contrast to wild type M1 RNA, which complements the ts mutation when it is overproduced, neither the C333 nor the U334 mutant M1 RNAs was able to do so.     

Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei.

RNAs that function in mitochondria are typically encoded by the mitochondrial DNA. However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion. It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import. We have identified and characterized mitochondrial precursor tRNAs in T. brucei. The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors. The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with [alpha-32P]CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P. We show that T. brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T. brucei tRNA precursors. Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis. The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.     

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.     

Recognition of the tRNA-like structure in tobacco mosaic viral RNA by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae

The 3'-terminal tRNA-like structure of the tobacco mosaic virus RNA interacts with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli or yeast in much the same manner as do tRNAs. Primary sites of interaction cluster near the 3' end and in the loop proposed to be analogous to the psi-loop of a tRNA. Some modified bases in the tRNA-like structure inhibit interaction with nucleotidyltransferase, yet the analogous bases in a tRNA do not. The location of some of these nucleotides within the analog to the psi-loop suggests that this structure differs slightly from its counterpart in a tRNA. The location of other such bases in the helical stem near the 3' end can be explained if the pseudoknot is disrupted by these modified bases or if the tertiary structure of the RNA is altered in the enzyme-RNA complex. A partially denatured secondary structure that persists on denaturing gels is proposed.     

Biosynthesis of transfer RNA: isolation and characterization of precursors to transfer RNA in the posterior silkgland of Bombyx mori.

Distinct low molecular weight RNA species that have properties expected for the precursor to tRNA have been isolated from the posterior silkglands of the silkworm Bombyx mori. These RNAs migrate between 4 S and 5 S markers on acrylamide gels and are labeled preferentially in vivo in relation to tRNA. The precursor RNAs can be converted specifically into molecules indistinguishable in size from tRNA upon incubation with “cleavage” enzymes isolated from the silkgland ribosomes. Two of the three low molecular weight RNAs contain the modified residues, pseudouridine, dihydrouridine and ribothymidine, and are methylated in vivo, suggesting that these base modifications occur while the tRNA is still in its precursor stage.     

Detection of long eukaryote-specific pyrimidine runs in repetitive DNA sequences and their relation to single-stranded regions in DNA isolated from sea urchin embryos.

Precursor molecules for Escherichia coli tRNAs that accumulated in a temperature-sensitive mutant defective in tRNA synthesis (TS709) were investigated. More than 20 precursors were purified by two-dimensional polyacrylamide gel electrophoresis. The purified molecules were analyzed by RNA fingerprint analysis and/or in vitro processing after treatment with E. coli cell-free extracts. The molecular sizes of most of the precursors identified were in the range of 4 to 5 S RNAs, although several larger ones were also detected. Fingerprint analysis revealed that the precursors generally differ from the corresponding mature tRNAs in the 5′ termini, having extra nucleotides. Thus, the genetic block in TS709 was shown to affect the trimming of the 5′ side of tRNA by impairing the function of RNAase P. Although this mutant had been isolated as a conditional mutant defective in the synthesis of su⁺ 3 tRNA₁^Tyr, the synthesis of many tRNA species was affected at high temperature. On the basis of their mode of maturation in vivo, the precursor molecules were discussed as intermediates in tRNA biosynthesis in E. coli. Accumulation of these intermediates was accounted for as a common feature of E. coli mutants defective in RNAase P function.     

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.     

Cytidines in tRNAs that are required intact by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae.

Individual species of tRNA from Escherichia coli were treated with hydrazine/3 M NaCl to modify cytidine residues. The chemically modified tRNAs were used as substrate for ATP/CTP: tRNA nucleotidyltransferases from E. coli and yeast, with [alpha-32P]ATP as cosubstrate. tRNAs that were labeled were analyzed for their content of modified cytidines. Cytidines at positions 74 and 75 were found to be required chemically intact for interaction with both enzymes. C56 was also required intact by the E. coli enzyme in all tRNAs, and by the yeast enzyme in several instances. C61 was found to be important in seven of 14 tRNAs with the E. coli enzyme but only in four of 13 tRNAs with that from yeast. Our results support a model in which nucleotidyltransferase extends from the 3' end of its tRNA substrate across the top of the stacked array of bases in the accepter- and psi-stems to the corner of the molecule where the D- and psi-loops are juxtaposed.     

Methylation of an adenosine in the D-loop of specific transfer RNAs from yeast by a procaryotic tRNA (adenine-1) methyltransferase.

tRNA (adenine-1) methyltransferase occurs in Bacillus subtilis. Eucaryotic tRNAThr and tRNATyr from yeast in which 1-methyladenosine (m1A) is already present in the TpsiC loop, can be methylated in vitro with S-adenosylmethionine and B. subtilis extracts. Each of the specific tRNAs accepts 1 mol of methyl groups per mol tRNA. The enzyme transforms into m1A the 3'-terminal adenylic acid residue of the dihydrouridine loop, a new position for a modified adenosine residue in tRNA. Both tRNAs have the sequence Py-A-A-G-G-C-m2(2)G in the D-loop and D-stem region. Other tRNAs with the same sequence in this region also serve as substrates for the tRNA (adenine-1) methyltransferase.     

Misacylation of tRNA with methionine in Saccharomyces cerevisiae

Accurate transfer RNA (tRNA) aminoacylation by aminoacyl-tRNA synthetases controls translational fidelity. Although tRNA synthetases are generally highly accurate, recent results show that the methionyl-tRNA synthetase (MetRS) is an exception. MetRS readily misacylates non-methionyl tRNAs at frequencies of up to 10% in mammalian cells; such mismethionylation may serve a beneficial role for cells to protect their own proteins against oxidative damage. The Escherichia coli MetRS mismethionylates two E. coli tRNA species in vitro, and these two tRNAs contain identity elements for mismethionylation. Here we investigate tRNA mismethionylation in Saccharomyces cerevisiae. tRNA mismethionylation occurs at a similar extent in vivo as in mammalian cells. Both cognate and mismethionylated tRNAs have similar turnover kinetics upon cycloheximide treatment. We identify specific arginine/lysine to methionine-substituted peptides in proteomic mass spectrometry, indicating that mismethionylated tRNAs are used in translation. The yeast MetRS is part of a complex containing the anchoring protein Arc1p and the glutamyl-tRNA synthetase (GluRS). The recombinant Arc1p–MetRS–GluRS complex binds and mismethionylates many tRNA species in vitro. Our results indicate that the yeast MetRS is responsible for extensive misacylation of non-methionyl tRNAs, and mismethionylation also occurs in this evolutionary branch.