Nuclear tRNA Research Articles

Role of the glycolytic protein, glyceraldehyde‐3‐phosphate dehydrogenase, in normal cell function and in cell pathology

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested. J. Cell. Biochem. 66:133-140, 1997. © 1997 Wiley-Liss, Inc.

Nucleotide sequence of nuclear tRNA(Gly) genes and tRNA(Gly) pseudogenes from yellow lupin (Lupinus luteus): expression of the tRNA(Gly) genes in vitro and in vivo.

A nuclear DNA fragment (7.8 kb) from yellow lupin (L. luteus) was sequenced and shown to contain tRNA(Gly) (GGC) genes and tRNAGly (GGC) pseudogenes organized in three tandemly repeated units: of 2565 bp and 2564 bp, and one, truncated from its 3' end, of 1212 bp. Each unit contains an identical pair of a tRNA(Gly) gene and a pseudogene, both having the same polarity. The nucleotide sequence of the gene appears colinear to L. luteus cytoplasmic tRNA(Gly) (GGC) primary structure. All three genes are efficiently transcribed in HeLa-cell nuclear extract giving two primary transcripts. The main, longer primary transcripts have each an extremely long 3' trailer of about 100 nucleotides, the structure of which is specific only for tRNAGly genes and pseudogenes (80% homology) of the studied tandem (but not for other tRNA(Gly) genes of the yellow lupin genome) as it has been shown by Southern hybridization. This distinctive feature allowed to isolate putative tRNAGly precursor(s) encoded by at least one of the three tRNA(Gly) (GGC) genes from L. luteus seedlings.

Distribution of both lengths and 5' terminal nucleotides of mammalian pre-tRNA 3' trailers reflects properties of 3' processing endoribonuclease.

Mammalian tRNA 3'processing endoribonuclease (3'tRNase) removes 3'extra nucleotides after the discriminator from tRNA precursors. Here I examined how the length of a 3'trailer and the nucleotides on each side of the cleavage site affected 3'processing efficiency. I performed in vitro 3'processing reactions of pre-tRNAArgs with various 3'trailers or various discriminator nucleotides using 3'tRNase purified from mouse FM3A cells or pig liver. On the whole, the efficiency of pre- tRNAArg3'processing by mammalian 3'tRNase decreased as the 3'trailer became longer, except in the case of a 3'trailer composed of CC, CCA or CCA plus 1 or 2 nucleotides, which was not able to be removed at all. The distribution of 3'trailer lengths deduced from mammalian nuclear tRNA genomic sequences reflects this property of 3'tRNase. The cleavage efficiency of pre-tRNAArgs varied depending on the 5'end nucleotide of a 3'trailer in the order G approximately A > U > C. This effect of the 5'end nucleotide was independent of the discriminator nucleotides. The distribution of the 5'end nucleotides of mammalian pre-tRNA 3'trailers reflects this differential 3'processing efficiency.

Nucleotide sequences of nuclear tRNA(Cys) genes from Nicotiana and Arabidopsis and expression in HeLa cell extract.

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA(GCA)Cys as a novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA(Cys) genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs(Cys). Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA(Cys) genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA(Cys) genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs(Cys) of plant origin.

Plant nuclear tRNA(Met) genes are ubiquitously interrupted by introns.

We have isolated three independent clones for nuclear elongator tRNA(Met) genes from an Arabidopsis DNA library using a tRNA(Met)-specific probe generated by PCR. Each of the coding sequences for tRNAMet in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA(Met) is dispersed at at least eight loci in the genome. The unspliced precursor tRNA(Met) intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA(Met) genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5'-distal end of each intron.

Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs.

The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7 transcript corresponding to a pre-tRNA(Tyr) from Arabidopsis thaliana has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3'-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5'-splice site. The reaction results in fragments with 2',3'-cyclic phosphate and 5'-OH termini. It is resistant to proteinase K and/or SDS treatment and is not inhibited by added tRNA. The self-cleavage depends on Mg2+ and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed for autolytic intron excision and, in parallel, for enzymatic in vitro splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than the enzymatic intron excision in a wheat germ S23 extract, we propose that the splicing endonuclease may function by improving the preciseness and efficiency of an inherent pre-tRNA self-cleavage activity.

Molecular cloning and sequencing of a nuclear gene encoding tRNA Gln (UUG) from Arabidopsis thaliana

A gene encoding cytoplasmic tRNA Gln (UUG) was isolated from an Arabidopsis DNA library. The coding sequence of the gene revealed 85% sequence identity with that of its animal counterparts. This is the first report of a nuclear tRNA Gln gene from higher plants.

The tRNA Tyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron-containing pre-tRNAs in wheat germ extract

Southern analysis of Triticum DNA has revealed that nuclear tRNA Tyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNA Tyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNA Tyr family. Four of the sequenced tRNA Tyr genes code for Triticum tRNA 1 Tyr and two code for tRNA 2 Tyr. Three genes encode tRNA Tyr which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNA Tyr genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5′ and 3′ splice site loops separated by five base pairs can be formed by all pre-tRNAs Tyr which are efficiently spliced in the homologous wheat germ extract.

Mechanisms Leading to and the Consequences of Altering the Normal Distribution of ATP(CTP):tRNA Nucleotidyltransferase in Yeast

CCA1 codes for mitochondrial, cytosolic, and nuclear ATP(CTP):tRNA nucleotidyltransferase. Studies reported here examine the mechanisms leading to and the consequences of altering the distribution of this important tRNA processing enzyme. We show that the majority of Cca1p-I, translated from the first in-frame ATG, is in mitochondria but surprisingly, there is a small contribution to nuclear and cytosolic tRNA processing by this form as well. The majority of Cca1p-II and Cca1p-III, translated from ATG2 and ATG3, respectively, is in the cytosol but both are also located in the nucleus for processing precursors. Altering the cytosolic/nuclear distribution of Cca1p by fusing the SV40 nuclear localization signal to the 5' end of CCA1 causes a growth defect and results in the accumulation of end-shortened tRNAs in the cytosol. These results suggest an important role for Cca1p in the cytosol of eukaryotes, presumably in the repair of 3' CCA termini. These experiments also demonstrate that individual tRNAs are affected differently by reduced cytosolic nucleotidyltransferase and that cells resuming exponential growth are more severely affected than those continuing exponential growth.

Mutation accumulation in transfer RNAs: molecular evidence for Muller's ratchet in mitochondrial genomes.

The accumulation of deleterious mutations is thought to be a major factor preventing the long-term persistence of obligately asexual lineages relative to their sexual ancestors. This phenomenon is also of potential relevance to sexual species that harbor asexually propagating organelle genomes. A comparative study of the transfer RNA genes in animal mitochondrial and nuclear genomes demonstrates that the former accumulate nucleotide substitutions much more rapidly than do the latter, and several lines of evidence are consistent with the idea that the excess substitutions are mildly deleterious. First, the average binding stability between complementary strands in the stems of mitochondrial tRNAs is less than half that in nuclear tRNAs. Second, most loop sizes in the mitochondrial tRNAs have experienced a net reduction in size over evolutionary time, and they are nearly 50 times more variable in the mitochondrial than in the nuclear genome. Third, although nearly 20% of the nucleotides in nuclear tRNA genes (particularly those involved in tertiary interactions) are invariant across all animal taxa and all tRNA species, there are no invariant sites in the mitochondrial tRNAs. These observations, as well as results from recent laboratory experiments, are consistent with the hypothesis that nonrecombining organelle genomes are subject to gradual loss of fitness due to the cumulative chance fixation of mildly deleterious mutations.

16] Isolation of mitochondrial tRNAs from human cells

This chapter explains the isolation of mitochondrial tRNAs from human cells. Mammalian mitochondrial tRNAs possess many unique sequence, structural, and functional properties. All mammalian mitochondrial DNAs (mtDNAs) code for 22 tRNAs, which are sufficient for the translation of the mtDNA-encoded mRNAs, because a single tRNA is able to read all codons of a four-codon family. In contrast to prokaryotic, eukaryotic nuclear, and chloroplast tRNAs, whose structures are remarkably conserved, there are numerous sequence and structural alterations found among mammalian mitochondrial tRNAs. These include changes in highly conserved nucleotides or nucleotide base modifications, variations in the sizes of stem and loop structures, and alterations in conserved tertiary interactions. The chapter describes procedures for the isolation of single tRNA species based on a variety of fractionation methods. tRNA fraction, isolated by diethylaminoethyl (DEAE) cellulose, is subsequently further fractionated by electrophoresis through successive polyacrylamide gels. In addition, the chapter also describes procedure for cultured mammalian cells that allows the isolation of a highly purified total mitochondrial tRNA preparation. This procedure has been used previously to investigate the steady-state levels and metabolic properties of tRNAs isolated from HeLa cell mitochondria.

Balanced translocation in a neuroblastoma patient disrupts a cluster of small nuclear RNA U1 and tRNA genes in chromosomal band 1p36.

Chromosomal band 1p36 probably harbours several neuroblastoma suppressor genes. A neuroblastoma patient has been described with a constitutional balanced translocation, t(1;17)(p36;q12-21). Cytogenetically, no loss of chromosomal material was visible. The 1p36 translocation breakpoint could therefore have inactivated one allele of a tumour suppressor gene, thus predisposing the patient to develop neuroblastoma. We localized this breakpoint by pulsed field gel electrophoresis, analysis of yeast artificial chromosomes, and fluorescence in situ hybridization. Here we report that the breakpoint is within a large cluster of small nuclear RNA U1 (RNU1) and some tRNA genes (TRE, TRN) on chromosomal band 1p36. The size of this cluster is over two megabases and it contains many other locally repeated sequences. Polyadenylated transcripts were identified for some of these sequences. In addition, the cluster is the target for integration of an adenovirus 5/SV40 hybrid virus. The translocation breakpoint maps distal of this viral integration site and proximal of marker PND.

Human Mitochondrial tRNA Processing

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.

Location of N 2, N 2-dimethylguanosine-specific tRNA methyltransferase

Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway. Indirect immunofluorescence experiments using antibody to N 2,N 2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells. Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p. Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease. Cells overproducing Trm1p also concentrate it to the nuclear periphery. Thus, the site(s) of interartion of Trm1p are not easily saturable and are likely to be in excess to Trm1p. Trm1p is shared by mitochondria and the nucleus. Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme. Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.

A nuclear tRNA gene cluster in the protozoan Leishmania tarentolae and differential distribution of nuclear-encoded tRNAs between the cytosol and mitochondria

All mitochondrial tRNAs in the protozoan Leishmania are believed to be encoded in the nuclear genome and imported selectively into the mitochondria by an as yet unknown mechanism. Previously, we reported that two tRNAs whose genes are tightly linked were imported by mitochondria. In contrast, a tRNA encoded by a lone tRNA gene was not detectable in mitochondria. The lone tRNA gene had flanking sequences that were different from the linked genes. These studies implied a possible correlation between tRNA gene organization and gene flanking sequence, and selective tRNA import into mitochondria. Here, we report the identification of a cluster of 10 tRNA genes and show the distribution of the corresponding tRNAs in cytosolic and mitochondrial fractions. tRNA leu(CAG) and tRNA 2 arg(TCG) are abundant in the cytosol, but relatively scarce in mitochondria. Conversely, tRNA ile(TAT) and tRNA 1 lys(TTT) are abundant in mitochondria, but relatively scarce in the cytosol, tRNA val(TAC) and tRNA 2 thr(TGT) are barely detectable in either cellular compartment, while tRNA gln(TTG), tRNA 1 arg(ACG), tRNA gly(TCC), and tRNA trp(CCA) are detected in approximately equal levels in both compartments. Sequencing of the 2600 bp that comprise the tRNA gene cluster also encoding the genes for 5S RNA and URNAB RNA indicates that nucleotide composition, length, and location of genes within the cluster do not clearly correlate with import characteristics. The unexpected presence of the tRNA trp(CCA)-gene transcript in mitochondria is also reported. Evidence suggests that this tRNA may have unidentified base modifications at the anticodon triplet.

In vivo expression and mitochondrial import of normal and mutated tRNA thr in Leishmania

Evidence suggests that mitochondria of protozoans and plants contain nuclear-encoded tRNAs. In trypanosomatids, the entire set of tRNAs in the mitochondria are presumably imported from the nucleus, but the mechanism of tRNA import is not presently understood. In this study, we have employed a plasmid-encoded nuclear tRNA gene as a means of investigating tRNA expression and mitochondrial import in vivo in Leishmania tarentolae. Using a Leishmania plasmid, we cloned a 1-kb or 250-bp restriction fragment carrying the nuclear tRNA thr gene and three in vitro mutagenized derivatives: Tac6 (an insertion of 6 nucleotides at the anticodon loop), Td4 (a 4-nt insert at the D-loop) and Tv4 (a 4-nt insert at the variable arm). Leishmania cells stably transfected with these plasmids were then examined for tRNA expression and import by Northern analysis. The results show that the plasmid-encoded wild type tRNA thr gene produced a significantly elevated level of expression in the cytosol. Similarly, the Tac6-transfected cells exhibited a large abundance of the mutant RNA relative to the normal tRNA (chromosome-encoded gene transcripts) in the cytosol. Furthermore, the mutant Tac6 RNA was found imported into mitochondria, although the proportion of the mutant vs. normal tRNA in mitochondria was greatly reduced as compared to that in the cytosol. We suggest that the mitochondrial import machinery is capable of discriminating against the mutant RNA in favor of the normal tRNA for import. In another example, we found that the Tv4 gene showed expression, albeit somewhat reduced, but its import into mitochondria was completely blocked. Unexpectedly, the 4-base addition mutation (Td4) at the D-loop showed neither expression nor import. While these results clearly signify the importance of various segments within the tRNA gene for in vivo expression, our data underscore the significance of the variable loop for mitochondrial import. It is our belief that this plasmid-encoded tRNA gene expression system in Leishmania may be useful in gaining further insights on tRNA import.

The tRNA(Ser)-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses.

The existence of six serine codons results in a complex pattern of tRNA(Ser) isoacceptors in organisms and organelles. According to the original wobble hypothesis, a minimum of three isoacceptors should be sufficient to read the six serine codons. We have isolated five cytoplasmic tRNAs(Ser) from leaves of Nicotiana rustica. Their nucleotide sequences identify them as four different isoacceptors with the anticodons cm5UGA, CGA, IGA and GCU. For tRNA(Ser) with IGA anticodon, two species have been detected which vary only by one nucleotide in the long extra arm. The first three isoacceptors recognize codons of the type UCN whereas the fourth isoacceptor reads the two serine codons AGC and AGU. The tRNA(Ser) sequences were used to design appropriate primers for the amplification of Nicotiana nuclear tRNA(Ser) genes by the polymerase chain reaction (PCR). A total number of eight tRNA(Ser) genes differing in the coding region were thus identified. Selected PCR DNA fragments were then employed as probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. Sequence analyses revealed that five of the isolated seven clones contained tRNA(Ser) genes which are identical in sequence with the five cytoplasmic tRNAs(Ser) mentioned above. None of them contains an intervening sequence. This is the first time that all putative cellular tRNA(Ser) isoacceptors and their corresponding genes have been characterized in an eukaryotic organism. Most of the tRNA(Ser) genes are functional as deduced from in vitro transcription and processing studies. Two of the genes yield pre-tRNAs(Ser) which are not or poorly converted to mature tRNA in a plant extract. The approximate tRNA(Ser) gene number was estimated by hybridization of specific DNA probes to Eco RI-cleaved Nicotiana nuclear DNA. The overall hybridization pattern indicates that members of each particular tRNA(Ser) gene family do not appear to be clustered but distributed randomly throughout the Nicotiana genome.

Identification of new eukaryotic tRNA genes in genomic DNA databases by a multistep weight matrix analysis of transcriptional control regions.

A linear method for the search of eukaryotic nuclear tRNA genes in DNA databases is described. Based on a modified version of the general weight matrix procedure, our algorithm relies on the recognition of two intragenic control regions known as A and B boxes, a transcription termination signal, and on the evaluation of the spacing between these elements. The scanning of the eukaryotic nuclear DNA database using this search algorithm correctly identified 933 of the 940 known tRNA genes (0.74% of false negatives). Thirty new potential tRNA genes were identified, and the transcriptional activity of two of them was directly verified by in vitro transcription. The total false positive rate of the algorithm was 0.014%. Structurally unusual tRNA genes, like those coding for selenocysteine tRNAs, could also be recognized using a set of rules concerning their specific properties, and one human gene coding for such tRNA was identified. Some of the newly identified tRNA genes were found in rather uncommon genomic positions: 2 in centromeric regions and 3 within introns. Furthermore, the presence of extragenically located B boxes in tRNA genes from various organisms could be detected through a specific subroutine of the standard search program.

Minimum intron requirements for tRNA splicing and nuclear transport in Xenopus oocytes

The presence or absence of an intron defines two classes of eukaryotic nuclear tRNA genes whose transcripts differ in a requirement for splicing. Using quantitative nuclear microinjection, we have previously found that nucleocytoplasmic transport of these two classes of tRNAs involves pathways which differ in one or more limiting components. To examine substrate features which distinguish these two pathways, a series of variants of a Xenopus tRNA(Tyr) gene were constructed in which the intron size was altered. The splicing and transport properties of the resulting transcripts were examined in oocyte microinjection and in vitro processing assays. The addition of one or two nucleotides at the splice site equivalent in an intronless gene produced transcripts which could be transported without splicing. However, transport was reduced relative to the mature-sequence tRNA, suggesting the anticodon loop (interrupted in pre-tRNAs) may be recognized by the intronless tRNA transport apparatus. Transcripts with four- or six-nucleotide intervening sequences were incompletely spliced with cleavage at only the 3' splice site. Neither unspliced precursor nor partially processed intermediates were efficiently transported. The results of coinjection experiments using tRNA and pre-tRNA competitors suggest that simple retention by the splicing apparatus may not account for failure to export these RNAs. Finally, a requirement for splicing is not unique to transport of pre-tRNA(Tyr) since a pre-tRNA(3Leu) variant which was not spliced was also not exported.

Endonucleolytic cleavage of a long 3'-trailer sequence in a nuclear yeast suppressor tRNA.

Transcripts of Saccharomyces cerevisiae nuclear tRNA genes are normally terminated within a few nucleotides of the tRNA coding region, in contrast to mitochondrially encoded tRNAs, which are contained within polycistronic transcripts and thus require 3'-processing by mitochondrial endonucleases. We show that 3'-processing activities capable of removing artificially extended 3'-trailer sequences from some tRNA substrates are also present in the yeast nucleus. Correct 3'-processing in vivo resulted in the formation of functional suppressor tRNA. The 3'-processing activities were also identified in vitro through analysis of transcription-processing products in cell-free yeast S-100 extracts. Comparison of several pre-tRNA substrates showed that the tRNA structure played a major role in determining the processability of a substrate but that the nature of the 3'-trailer sequence also modulated the rate of 3'-processing. Pre-tRNA containing mitochondrial tRNA(Val) sequence was a good substrate for in vitro processing, independent of its 3'-trailer. A 200-nt-long pre-tRNA, encoding the nuclear SUP4 tRNA gene and a mitochondrial 3'-trailer, was processed in yeast S-100 extract in a multistep pathway into mature-sized tRNA(Tyr). Part of the 3'-processing was due to an endonuclease which cleaved near or precisely at the 3'-end of the coding region of the tRNA. A short sequence around this endonucleolytic 3'-cleavage site was crucial for the formation of active suppressor tRNA in vivo. A 9-nt-long sequence motif derived from the mitochondrial 3'-trailer allowed processing, while sequences derived from lacZ or pBR322 DNA were processed neither in vitro nor in vivo.