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Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies-purified and extract-based systems-and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The essential and universal N(6)-threonylcarbamoyladenosine (t(6)A) modification at position 37 of ANN-decoding tRNAs plays a pivotal role in translational fidelity through enhancement of the cognate codon recognition and stabilization of the codon-anticodon interaction. In Escherichia coli, the YgjD (TsaD), YeaZ (TsaB), YjeE (TsaE) and YrdC (TsaC) proteins are necessary and sufficient for the in vitro biosynthesis of t(6)A, using tRNA, ATP, L-threonine and bicarbonate as substrates. YrdC synthesizes the short-lived L-threonylcarbamoyladenylate (TCA), and YgjD, YeaZ and YjeE cooperate to transfer the L-threonylcarbamoyl-moiety from TCA onto adenosine at position 37 of substrate tRNA. We determined the crystal structure of the heterodimer YgjD-YeaZ at 2.3 Å, revealing the presence of an unexpected molecule of ADP bound at an atypical site situated at the YgjD-YeaZ interface. We further showed that the ATPase activity of YjeE is strongly activated by the YgjD-YeaZ heterodimer. We established by binding experiments and SAXS data analysis that YgjD-YeaZ and YjeE form a compact ternary complex only in presence of ATP. The formation of the ternary YgjD-YeaZ-YjeE complex is required for the in vitro biosynthesis of t(6)A but not its ATPase activity., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3'-terminus (N+1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30,000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3'-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3'-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

How eukaryotic RNA polymerases switch from elongation to termination is unknown. Pol III subunits Rpc53 and Rpc37 (C53/37) form a heterodimer homologous to TFIIFβ/α. C53/37 promotes efficient termination and together with C11 also mediates pol III recycling in vitro. We previously developed Schizosaccharomyces pombe strains that report on two pol III termination activities: RNA oligo(U) 3'-end cleavage, and terminator readthrough. We randomly mutagenized C53 and C37 and isolated many C37 mutants with terminator readthrough but no comparable C53 mutants. The majority of C37 mutants have strong phenotypes with up to 40% readthrough and map to a C-terminal tract previously localized near Rpc2p in the pol III active center while a minority represent a distinct class with weaker phenotype, less readthrough and 3'-oligo(U) lengthening. Nascent pre-tRNAs released from a terminator by C37 mutants have shorter 3'-oligo(U) tracts than in cleavage-deficient C11 double mutants indicating RNA 3'-end cleavage during termination. We asked whether termination deficiency affects transcription output in the mutants in vivo both by monitoring intron-containing nascent transcript levels and (14)C-uridine incorporation. Surprisingly, multiple termination mutants have no decrease in transcript output relative to controls. These data are discussed in context of current models of pol III transcription.

Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA(i)(Met) and other tRNA precursors, and the aminoacylation of tRNA(i)(Met) are also strongly impaired in gua1-G388D cells, tRNA(i)(Met)-containing complexes with the macromolecular composition of the eIF2·tRNA(i)(Met.)GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate ∼10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Δhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.

Dr1 (also known as NC2beta) was identified as a repressor of RNA polymerase (pol) II transcription. It was subsequently shown to inhibit pol III transcription when expressed at high levels in vitro or in yeast cells. However, endogenous Dr1 was not detected at pol III-transcribed genes in growing yeast. In contrast, we demonstrate that endogenous Dr1 is present at pol III templates in human cells, as is its dimerization partner DRAP1 (also called NC2alpha). Expression of tRNA by pol III is selectively enhanced by RNAi-mediated depletion of endogenous human Dr1, but we found no evidence that DRAP1 influences pol III output in vivo. A stable association was detected between endogenous Dr1 and the pol III-specific transcription factor Brf1. This interaction may recruit Dr1 to pol III templates in vivo, as crosslinking to these sites increases following Brf1 induction. On the basis of these data, we conclude that the physiological functions of human Dr1 include regulation of pol III transcription.

Ribonuclease P (RNase P) has been hitherto well known as a catalytic ribonucleoprotein that processes the 5' leader sequence of precursor tRNA. Recent studies, however, reveal a new role for nuclear forms of RNase P in the transcription of tRNA genes by RNA polymerase (pol) III, thus linking transcription with processing in the regulation of tRNA gene expression. However, RNase P is also essential for the transcription of other small noncoding RNA genes, whose precursor transcripts are not recognized as substrates for this holoenzyme. Accordingly, RNase P can act solely as a transcription factor for pol III, a role that seems to be conserved in eukarya.

TFIIIC is a RNA polymerase (pol) III-specific DNA-binding factor that is required for transcription of tRNA and 5S rRNA genes. Active human TFIIIC consists of five subunits. However, an inactive form has also been isolated that lacks one of the five subunits, called TFIIIC110. A model was proposed in which pol III transcription might be regulated by the specific induction of TFIIIC110, allowing formation of active TFIIIC from the inactive form. We have tested this model by transient transfection of HeLa and HEK293 cells with a vector expressing TFIIIC110. We have also made stably transfected HeLa cell lines that carry a doxycycline-inducible version of the cDNA for TFIIIC110. We show that the induced TFIIIC110 enters the nucleus, binds other TFIIIC subunits and is recruited to tRNA and 5S rRNA genes in vivo. However, little or no effect is seen on the expression of pol III transcripts. The data argue against the model that pol III transcription can be effectively modulated through the specific induction of TFIIIC110.

Despite the ubiquitous distribution of tRNA-related short interspersed elements (SINEs) in eukaryotic species, very little is known about the synthesis and processing of their RNAs. In this work, we have characterized in detail the different RNA populations resulting from the expression of a tRNA-related SINE S1 founder copy in Arabidopsis thaliana. The main population is composed of poly(A)-ending (pa) SINE RNAs, while two minor populations correspond to full-length (fl) or poly(A) minus [small cytoplasmic (sc)] SINE RNAs. Part of the poly(A) minus RNAs is modified by 3'-terminal addition of C or CA nucleotides. All three RNA populations accumulate in the cytoplasm. Using a mutagenesis approach, we show that the poly(A) region and the 3' end unique region, present at the founder locus, are both important for the maturation and the steady-state accumulation of the different S1 RNA populations. The observation that primary SINE transcripts can be post-transcriptionally processed in vivo into a poly(A)-ending species introduces the possibility that this paRNA is used as a retroposition intermediate.

The updated version of PLMItRNA reports information and multialignments on 609 genes and 34 tRNA molecules active in the mitochondria of Viridiplantae (27 Embryophyta and 10 Chlorophyta), and photosynthetic algae (one Cryptophyta, four Rhodophyta and two Stramenopiles). Colour-code based tables reporting the different genetic origin of identified genes allow hyper-textual link to single entries. Promoter sequences identified for tRNA genes in the mitochondrial genomes of Angiospermae are also reported. The PLMItRNA database is accessible at http://bighost.area.ba.cnr.it/PLMItRNA/.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

tRNA isopentenylation is a branch of an isoprenoid pathway in yeast. There is a competition for a substrate between isoprenoid biosynthetic enzyme Erg20p and tRNA isopentenyltransferase. Here we studied the direct effect of elevated tRNA biosynthesis on ERG20 expression. The maf1-1 mutant of Saccharomyces cerevisiae that has enhanced cellular tRNA levels was used. We show that both ERG20 transcript and Erg20 protein levels are increased in maf1-1. Additionally, maf1-1 leads to decreased ergosterol content in the cells. These effects of maf1-1 are dependent on functional tRNA isopentenyltransferase. Our results indicate that a complex regulation of the isoprenoid pathway involves also an effect of changes in tRNA biosynthesis.

Acetaldehyde is present in tobacco smoke and automotive exhaust gases, is produced by the oxidation of ethanol, and causes respiratory organ cancers in animals. We show both the types and spectra of acetaldehyde-induced mutations in supF genes in double- and single-stranded shuttle vector plasmids replicated in human cells. Of the 101 mutants obtained from the double-stranded plasmids, 63% had tandem base substitutions, of which the predominant type is GG to TT transversions. Of the 44 mutants obtained from the single-stranded plasmids, 39% had tandem mutations that are of a different type than the double-stranded ones. The GG to TT tandem substitutions could arise from intra-strand crosslinks. Our data indicate that acetaldehyde forms intra- as well as inter-strand crosslinks between adjacent two-guanine bases. Based upon the following observations: XP-A protein binds to acetaldehyde-treated DNA, DNA excision repair-deficient xeroderma pigmentosum (XP) cells were more sensitive to acetaldehyde than the repair-proficient normal cells, and a higher frequency of acetaldehyde-induced mutations of the shuttle vectors was found in XP cells than in normal cells, we propose that the DNA damage caused by acetaldehyde is removed by the nucleotide excision repair pathway. Since treatment with acetaldehyde yields very specific GG to TT tandem base substitutions in DNA, such changes can be used as a probe to identify acetaldehyde as the causal agent in human tumors.

Glyoxal is a major product of DNA oxidation in which Fenton-type oxygen free radical-forming systems are involved. To determine the mutation spectrum of glyoxal in mammalian cells and to compare the spectrum with those observed in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA gene (supF) in the shuttle vector plasmid pMY189. We treated pMY189 with glyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and mutation frequency increased according to the dose of glyoxal. The majority of glyoxal-induced mutations (48%) were single-base substitutions. Eighty three percent of the single-base substitutions occurred at G:C base pairs. Among them, G:C-->T:A transversions were predominant, followed by G:C-->C:G transversions and G:C-->A:T transitions. A:T-->T:A transversions were also observed. Mutational hotspots within the supF gene were detected. These results suggest that glyoxal may play an important role in mutagenesis induced by oxygen free radicals.

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

The tufB gene, encoding elongation factor Tu (EF-Tu), from the myxobacterium Stigmatella aurantiaca was cloned and sequenced. It is preceded by four tRNA genes, the first ever described in myxobacteria. The tRNA synthesized from these genes and the general organization of the locus seem identical to that of Escherichia coli, but differences of potential importance were found in the tRNA sequences and in the intergenic regions. The primary structure of EF-Tu was deduced from the tufB DNA sequence. The factor is composed of 396 amino acids, with a predicted molecular mass of 43.4 kDa, which was confirmed by expression of tufB in maxicells. Sequence comparisons between S.aurantiaca EF-Tu and other bacterial homologues from E.coli, Salmonella typhimurium and Thermus thermophilus displayed extensive homologies (75.9%). Among the variable positions, two Cys residues probably involved in the temperature sensitivity of E.coli and S.typhimurium EF-Tu are replaced in T.thermophilus and S.aurantiaca EF-Tu. Since two or even three tuf genes have been described in other bacterial species, the presence of multiple tuf genes was sought for. Southern and Northern analysis are consistent with two tuf genes in the genome of S.aurantiaca. Primer extension experiments indicate that the four tRNA genes and tufB are organized in a single operon.

Peptidyl-transfer RNA normally dissociates at a low rate from the ribosomes of Escherichia coli during protein synthesis but accumulates under nonpermissive conditions in cells with a temperature-sensitive allele (pthts) of the gene encoding peptidyl-transfer RNA hydrolase. The antibiotic-hypersensitive strain E. coli DB-11 with the pthts mutation was exposed to viomycin, then placed at nonpermissive temperatures. Under these conditions in the absence of drugs, peptidyl-tRNA accumulates, protein synthesis is inhibited and pthts cells die. When viomycin was present at sufficient concentration to arrest protein synthesis, cell death was not accelerated, error-inducing effects of streptomycin were not counteracted and, at high doses, cytoplasmic accumulation of peptidyl-transfer RNA was slowed down. Blocking the translocation of peptidyl-transfer RNA with viomycin did not stimulate its dissociation from ribosomes. Erythromycin-enhanced cell death was not affected by viomycin at doses sufficient to block amino acid incorporation, suggesting that short peptidyl-transfer RNAs could still be synthesized and dissociated from ribosomes.

The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNA(ser)(AGN), which is typical of metazoan mtDNAs, and also in tRNA(ser)(UCN), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA(glu) and CO3 contains a 72-bp tract of alternating A and T.

In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E. coli strain that produces saturating amounts of T7 RNA polymerase. At 37 degrees C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E. coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript.

Three recombinant plasmids containing randomly sheared genomic D. melanogaster tRNAs have been identified and characterized in detail. One of these, the plasmid 14C4, has a D. melanogaster (Dm) DNA segment of 18 kb, and has three tRNA2Arg and two tRNAAsN genes. The second plasmid, 38B10, has tRNAHis genes, while the third plasmid, 63H5, contains coding sequences for tRNA2Asp. The Dm DNA segments in each recombinant plasmid are derived from unique cytogenetic loci. 14C4 is from 84 F, 38B10 is from 48 F and 63H5 is from 70 A.

The distal region of the Escherichia coli ribosomal RNA operon rrnD has been sequenced to identify transfer RNA genes which might be contained within this transcriptional unit, and to define a possible transcription termination site. A nucleotide sequence corresponding to the gene for tRNA1thr is located between two genes for 5S rRNA. The spacer region between the proximal 5S rRNA gene and the tRNA1thr gene is 12 base pairs long, whereas the spacer region between the distal 5S rRNA gene and the tRNA1thr gene is 37 base pairs. Possible sites for RNA processing are discussed, and the sequence of the tRNA1thr gene is found to possess the same anticodon as the tRNA3thr gene. A putative transcription termination site for rrnD was located about 25 base pairs distal to the 3' end of the second 5S rRNA gene. The rrnD and rrnC terminators are compared with each other and with other prokaryotic terminators. Located at exactly the same site on the complementary strand is another putative transcription termination site for a gene being transcribed towards rrnD. The possibility of overlapping terminators is discussed.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.

Transfer RNA's were isolated from Euglena gracilis. Chloroplast cistrons for tRNA were quantitated by hybridizing tRNA to ct DNA. Species of tRNA hybridizing to ct DNA were partially purified by hybridization-chromatography. The tRNA's hybridizing to ct DNA and nuclear DNA appear to be different. Total cellular tRNA was hybridized to ct DNA to an equivalent of approximately 25 cistrons. The total cellular tRNA was also separated into 2 fractions by chromatography on dihydroxyboryl substituted amino ethyl cellulose. Fraction I hybridized to both nuclear and ct DNA. Hybridizations to ct DNA indicated approximately 18 cistrons. Fraction II-tRNA hybridized only to ct DNA, saturating at a level of approximately 7 cistrons. The tRNA from isolated chloroplasts hybridized to both chloroplast and nuclear DNA. The level of hybridization to ct DNA indicated approximately 18 cistrons. Fraction II-type tRNA could not be detected in the isolated chloroplasts.

The Q nucleosides isolated from rabbit liver tRNA are known to have sugars (mannose or galactose) linked to their cyclopentene diol moiety. A Q nucleoside containing mannose (manQ) was synthesized by a cell-free system from rat liver, using purified E. coli tRNAAsp as an acceptor and GDP-mannose as a donor molecule. The novel mannosyltransferase catalyzing this reaction was purified from a particulate-free soluble enzyme fraction and found to be strictly specific for tRNAAsp. These results, together with the anomeric configuration of mannose in Q nucleoside, indicate that no lipid intermediate is involved in the biosynthesis of Q nucleoside.

In vivo yeast precursor tRNAs have been identified using a modification of the Northern-hybridization procedure. Two species of pre-tRNA Tyr, 1 species of pre-tRNA Ser2 and 2 species of pre-tRNA Serminor have been found in all yeast strains examined, including parental strains and strains harboring mutations affecting tRNA function. One of the tRNA Tyr strains harboring and one of the pre-tRNA SerUCG are the same size as the unspliced pre-tRNAs which accumulate in the yeast mutant rna1. The in vivo tRNA Tyr precursors detected in these studies also appear similar with the RNA species identified when cloned yeast tRNA Tyr is transcribed and processed by Xenopus oocytes and/or Xenopus extracts. We have also studied the precursor and mature tRNA Tyr species from 22 mutants which contain mutations in the SUP4 tyrosine-inserting suppressor locus. The RNA from 2 mutants mapping at the G52 position showing an aberrantly migrating "mature" tRNA Tyr. Although several of those cloned mutant genes showed transcript products of altered size in in vitro transcription studies (1), we did not detect such altered transcripts in vivo.

The antibiotic rifampicin, which in prokaryotes inhibits the initiation of RNA synthesis but not the completion of nascent strands, was used to explore tRNA gene transcriptional organization in Escherichia coli. Cultures were grown in [32P] orthophosphate to constant specific radioactivity and labeled with [3H] uridine in the presence of rifampicin. Numerous tRNA species then were isolated by polyacrylamide gel electrophoresis and their 3H/32P ratios determined; these ratios, following correction for the base compositions of the tRNAs, should reflect the distances of the corresponding tRNA genes from their promoters. Individual tRNA species were identified, where possible, by oligonucleotide fingerprint analysis. Observed isotopic ratios were correlated with promoter-gene distances, measured in nucleotides, using the nucleotide sequence of the 16S ribosomal RNA gene as a reference. The protocols developed should be applicable to most prokaryotes.

The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N(6)-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N(6)-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N(6)-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3'-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N(6)-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N(6)-dimethyladenosine on the tRNA level in the same organ is discussed.

Yeast mitochondrial DNA contains a genetic locus, called the tRNA synthesis locus, which codes for information necessary for mitochondrial tRNA biosynthesis. A 9S RNA molecule coded by this locus is thought to be the trans-acting element required for the removal of 5' extensions from tRNA precursors. The DNA coding for this RNA maps to a region of mitochondrial DNA known to contain strain specific restriction site polymorphisms. Comparison of the tRNA synthesis locus in two such strains by sequence analysis demonstrates that the restriction enzyme polymorphisms are due to the deletion/insertion of a 50 base pair GC-rich element in the 5' flanking sequence of the 9S RNA coding region. There are also several differences between the 9S RNA coding region of these two strains which do not interfere with the tRNA synthesis function.

Expression of 5 yeast mitochondrial tRNA genes (Ala, Ile, Tyr, Asn and Metm), localized upstream from the oxil gene has been analyzed by in vitro capping using guanylyltransferase, northern hybridization and S1 nuclease mapping in the wild type and a rho-strain. The 5 tRNA sequences belong to the same transcriptional unit which is initiated 133 bp upstream from the tRNA(Ala) gene at a promoter sequence TTATAAGTA. Furthermore, a truncated tRNA(Tyr) transcript, 2 nucleotides shorter than mature tRNA(Tyr) has been found, only in the rho-strain. This minor transcript may result from secondary transcription initiation at a variant nonanucleotide sequence, ATATAAGGA, which overlaps the tRNA(Tyr) coding sequence by 3 nucleotides. The polycistronic precursor has proven to be useful in investigation of the mechanisms of tRNA processing. Maturation of this primary transcript proceeds exclusively by precise endonucleolytic cleavages at the 5' and 3'-ends of tRNA sequences.

1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed.

Analysis of primary transcripts made by Escherichia coli RNA polymerase on T4 DNA containing an intact or partially deleted tRNA gene cluster demonstrates that the T4 tRNA genes are transcribed from two promoters differing in their strength. The stronger (P1) and the weaker (P2) promoters are located at distances of 1 kb and 1.5 kb from the tRNA genes, respectively. Selective initiation of individual transcripts with dinucleotides shows that P1 and P2 promoters contain the sequences TAT and CAC respectively. The two-promoter organisation of the tRNA cluster may reflect two superimposed mechanisms of gene expression in T4-infected bacteria.

Nucleotide pyrophosphate transferase isolated from Streptomyces griseus is used to transfer pyrophosphate group from gamma-32P-ATP to the 3'-OH of tRNA, generating a strictly terminal label at its 3' end. Using yeast tRNAPhe as model compound, it is demonstrated that the labelled molecule is suitable for rapid gel sequencing by both enzymatic and chemical methods. RNA molecules terminated by pyrimidine nucleoside are poor pyrophosphate acceptors. To label RNAs of this kind, first guanosine 5'-phosphate 3'-(beta-32P)-pyrophosphate (pGpp) is prepared from gamma-32P-ATP and GMP by nucleotide pyrophosphate transferase. pGpp is then ligated to the 3' end of RNA by T4 RNA ligase. The complete nucleotide sequence of 5S RNA from Streptomyces griseus is established by rapid gel sequencing methods performed on 3'-(beta-32P)-pyrophosphate labelled molecule.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.

The nucleotide sequence of a low molecular weight RNA coded by bacteriophage T4 (and previously identified as species alpha) has been determined. The molecule is of particular biological interest for its associated biosynthetic properties. This RNA is 76 nucleotides in length, contains eight modified bases, and can be arranged in a cloverleaf configuration common to tRNAs. The anticodon sequence is UGU, which corresponds to the threonine-specific codons ACA G. The nucleotide sequence was determined primarily by nearest-neighbor analysis of RNA synthesized in vitro using [alpha-32P]nucleoside triphosphates. Using the single-strand specific nuclease S1, two in vivo labeled half-molecules were generated and analysed. This information together with restrictions imposed by nearest-neighbor data, provided a unique linear sequence of nucleotides with the features of secondary structure common to tRNA molecules.

Treatment of the ciliated protozoan Tetrahymena thermophila with 8-methoxypsoralen combined with long wavelength ultraviolet irradiation (UVA, lambda approximately 360 nm) resulted in a dose dependent equal inhibition of the synthesis of rRNA, 5.8S, 5S and tRNA. Similar results were obtained with 3-carbethoxy-8-methoxypsoralen which predominantly forms DNA mono-adducts. In contrast the synthesis of tRNA in T. thermophila was much less sensitive than that of rRNA, 5.8S and 5S RNA to treatment with short wavelength ultraviolet irradiation (UVB, lambda approximately 254 nm). These results are interpreted in favor of a mechanism by which psoralen-DNA adducts (crosslinks much greater than monoadducts) inhibit RNA transcription initiation (in contrast to UVB which causes premature chain termination). Furthermore it is argued that RNA synthesis is regulated in equally sized domains regardless of the gene-size.

HisU mutants of Salmonella typhimurium are depressed in the histidine operon since they have lower intracellular concentration of histidyl-tRNAHis. In this paper we present evidences showing that a strain carrying a hisU mutation (hisUl206) is altered in a nucleolytic enzyme involved in tRNA maturation process. The analysis of several hisU mutants indicates that hisU region of bacterial genome may account for more than one function involved in tRNA biosynthesis.

In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable. In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant. When the mutant was grown in the absence of the drug, the resistance was rapidly lost. This character showed a typical cytoplasmic inheritance. The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA. The amplified molecules were co-replicating with the wild type genome in the mutant cells. The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA. The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl). Modified processing of these tRNAs was detected in the mutant. A possible mechanism by which these events could lead to drug resistance is discussed.

Several DNA fragments carrying tRNA genes have been cloned from EcoRI endonuclease digests of Escherichia coli DNA. Using cloned DNA, the sequence of the region around the distal gene for tRNA1Asp (F(or G)) in the E. coli ribosomal RNA operon [rrnF(or G)] has been determined. In the distal portion of rrnF(or G), the genes for 23S, 5S rRNA and tRNA1Asp (F(or G)) are located in that order and separated by intergenic spacers of 93 and 52 base pairs, respectively. A possible hairpin structure, with its center between the 22nd and 23rd base pair downstream from the 3'-end of the tRNA1Asp(F(or G)) gene, followed by a sequence of eight thymidine residues was identified as the transcription termination signal for rrnF(or G). The termination is rho-independent, at least in vitro, and occurs within the region of the contiguous thymidine residues. A possible promoter for a protein gene is present about 50 base pairs downstream from the rrnF(or G) terminator.

A tetradecanucleotide U-A-G-C(U-C-G)2G-G-C-Up corresponding to bases 21-34 of a nascent sequence of formylmethionyl tRNA of E. coli has been synthesized by the joining of two combinations of chemically synthesized oligonucleotides: 1) U-A-G-C + U-C-G-U-C-G + G-G-C-Up and 2) U-A-G-C + U-C-G-U + C-G-G-G-C-Up. In reaction 1) and the extent of joining *pG-G-C-Up to U-C-G-U-C-G was only 15.4% and the last ligation of the decamer to U-A-G-U proceeded to 27%. In reaction 2) joining between U-A-G-C and pU-C-G-Up gave a high yield (88%). The ligation of this octamer and *pC-G-G-G-C-Up also gave a satisfactory yield (52%). These reactions suggest that sequence preferences in RNA ligase reactions may arise from the structure of the 3'-end of acceptor molecules.

Formycin, an adenosine analog, was used in studies of RNA synthesis by silk glands of silkworms in organ culture. Formycin has been shown to inhibit preferentially the incorporation of [3H]uridine into low molecular weight RNA in the cytoplasm. When silk glands were pulse-chase labeled with [3H]uridine in the presence of formycin, the synthesis of 4.5S tRNA precursors was not affected, as determined by gel electrophoresis. However, the accumulation of mature 4S tRNA was greatly reduced in the formycin-treated system, although the precursors synthesized disappeared from the 4.5S region on chase at the same rate as in the control system. [14C]Formycin was incorporated into the 4.5S precursors but not into 4S tRNA. These findings suggest that the inhibition of tRNA synthesis by formycin is due to the existence of some degradation mechanisms of formycin-containing 4.5S precursor RNA's and the failure to process them into normal 4S tRNA.