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In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

Streptococcus pneumoniae is a causative agent of nosocomial infections such as pneumonia, meningitis, and septicemia. Penicillin resistance in S. pneumoniae depends in part upon MurM, an aminoacyl-tRNA ligase that attaches L-serine or L-alanine to the stem peptide lysine of Lipid II in cell wall peptidoglycan. To investigate the exact substrates the translation machinery provides MurM, quality control by alanyl-tRNA synthetase (AlaRS) was investigated. AlaRS mischarged serine and glycine to tRNA(Ala), as observed in other bacteria, and also transferred alanine, serine, and glycine to tRNA(Phe). S. pneumoniae tRNA(Phe) has an unusual U4:C69 mismatch in its acceptor stem that prevents editing by phenylalanyl-tRNA synthetase (PheRS), leading to the accumulation of misaminoacylated tRNAs that could serve as substrates for translation or for MurM. Although the peptidoglycan layer of S. pneumoniae tolerates a combination of both branched and linear muropeptides, deletion of MurM results in a reversion to penicillin sensitivity in strains that were previously resistant. However, because MurM is not required for cell viability, the reason for its functional conservation across all strains of S. pneumoniae has remained elusive. We now show that MurM can directly function in translation quality control by acting as a broad specificity lipid-independent trans editing factor that deacylates tRNA. This activity of MurM does not require the presence of its second substrate, Lipid II, and can functionally substitute for the activity of widely conserved editing domain homologues of AlaRS, termed AlaXPs proteins, which are themselves absent from S. pneumoniae.

4-Thiouridine (s(4)U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s(4)U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s(4)U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s(4)U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s(4)U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s(4)U. In the absence of an RLD, MMP1354 uses Cys(265) and Cys(268) located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S(2-) is a physiologically relevant sulfur donor for s(4)U formation catalyzed by MMP1354 (K(m) for Na(2)S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s(4)U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.

Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

The c-Myc oncoprotein promotes cell growth by enhancing ribosomal biogenesis. Overexpression of c-Myc and aberrant ribosomal biogenesis lead to deregulated cell growth and tumorigenesis. Hence, c-Myc activity and ribosomal biogenesis must be tightly coordinated during normal homeostasis. We previously found that ribosomal protein L11 inhibits c-Myc activity by blocking the recruitment of its co-activator transformation/transcription domain-associated protein (TRRAP) to the promoter regions of c-Myc target genes that are transcribed by RNA polymerases I and II. In this study, we extended the role of L11 to the regulation of c-Myc-driven transcription of the 5 S rRNA and tRNA genes by RNA polymerase III. L11 co-resided with c-Myc at the 5 S rRNA and tRNA genes and significantly inhibited the binding of TRRAP to these genes. Knocking down endogenous L11 enhanced c-Myc-dependent transcription of these genes. Interestingly, in response to ribosomal stress induced by the treatment of cells with a low dose of actinomycin D or serum starvation, L11 binding to these genes was increased, and inversely TRRAP binding to these genes was decreased. Consistently, knockdown of L11 rescued the reduction of the expression of these genes by the two treatments. These results demonstrate that L11 suppresses c-Myc-dependent and RNA polymerase III-catalyzed transcription of 5 S rRNA and tRNA genes in response to ribosomal stress, ensuring a tight coordination between c-Myc activity and ribosomal biogenesis.

The chloroplast genome of a dinoflagellate consists of a group of small circular DNA molecules (minicircles), most of which carry a single gene. With RT-PCR, primer extension, and Northern analyses, we show that the entire minicircle is transcribed and that some minicircles can produce RNAs larger than themselves. Using an RNA ligase-mediated rapid amplification of cDNA ends method, we were able to detect large processed precursors that are generated by endonucleolytic cleavage of an even longer molecule. This cleavage produces the mature mRNA 3'-end and at the same time the 5'-end of the precursor. The tRNAs encoded by the petD and psbE minicircles appear to be processed in the same way. We propose a "rolling circle" model for chloroplast transcription in which transcription would proceed continuously around the minicircular DNA to produce transcripts larger than the minicircle itself. These transcripts would be further processed into discrete mature mRNAs and tRNAs.

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded and imported into the mitochondrion. A heterogeneous population of RNAs having characteristics of precursor tRNAs have previously been identified within the mitochondrion of T. brucei, suggesting that import occurs via a precursor molecule. In order to identify nuclear genes encoding tRNAs targeted to the mitochondrion, individual mitochondrial tRNAs were separated using two-dimensional gel electrophoresis and enzymatically sequenced. A 1.1-kilobase pair genomic DNA fragment was cloned containing three tRNA genes, tRNA(1)(Ser), tRNA(Leu), and tRNA(2)(Ser). Dicistronic precursors containing the tRNA(1)(Ser) and tRNA(Leu) transcripts with a 59-nucleotide intergenic sequence were identified by reverse transcriptase and polymerase chain reactions and the 5' end of the precursors determined. The dicistronic precursor tRNA is present both in the cytosol and the mitochondrion supporting a model for tRNA import involving precursor tRNA transcripts.

Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.

We report the characterization of a mutation affecting tau 138, the largest subunit of yeast transcription factor IIIC (TFIIIC). A previously described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S.D., and Pringle, J.R. (1991) Genetics 127, 279-285) is shown to lie in the TFC3 gene which encodes tau 138. The tau 138 subunit carrying this mutation bears a single substitution of Glu for Gly at position 349 (G349E). In extracts from mutant cells, both the level of TFIIIC and its affinity for tDNA were found to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel shift assays. In addition, the tsv115 mutation affected 5 S RNA synthesis in vitro, suggesting that the tau 138 subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of the TFIIIC defect were sought to reveal components participating in TFIIIC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau 95 and tau 131, the 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymerase (pol) I, II, and III, ABC10 alpha; it also includes genes potentially related to pol III function, such as SRP40 which also suppresses a mutation in a subunit shared by RNA polymerases I and III. A second class of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.

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.

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.

1. Normal mouse liver contains predominantly tRNA that contains queuosine in the first position of the anticodon ((Q+)tRNA). 2. Germ-free mice fed a chemically defined diet devoid of queuine for 1 year have no queuine in all four of the tRNAs that respond to the NAUC codons. 3. The synthesis of (Q+)tRNAs can be induced by injecting queuine, feeding free queuine, or by feeding (Q+)tRNA. 4. When mice that have no (Q+)tRNA are titrated with exogenous queuine, tRNAAsp is modified to the (Q+) state before tRNAHis.

Cell-free extracts (S-100) prepared from cultured mammalian KB cells have previously been shown to direct accurate and selective transcription of class III genes by RNA polymerase III. We have fractionated the KB S-100 and have found that multiple components are essential for the accurate transcription of these genes. After the S-100 has been separated into four different protein fractions by chromatography on phosphocellulose, two fractions are required, in addition to RNA polymerase III, for active and selective transcription of the virus-associated RNAI gene of adenovirus 2 and a tRNA gene; a third fraction is required, along with these components, for the reconstitution of 5 S RNA gene transcription. At least two of these components are distinct from the four factors required for accurate initiation of transcription by RNA polymerase II (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. Biol. Chem. 255, 11992-11996).

Chemical syntheses of the tridecanucleotide, d(G-C-T-T-C-C-C-G-A-T-A-A-G), the dodecanucleotide, d(G-C-T-C-C-C-T-T-A-T-C-G), the decanucleotide, d(G-G-A-G-C-A-G-G-C-C), the nonanucleotide, d(T-A-C-T-G-G-C-C-T), and the hexanucleotide, d(G-G-A-A-G-C), are described. Together, these syntheses represent the nucleotide sequence 100-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor. The synthesis of the dodecanucleotide d(G-C-T-C-C-C-T-T-A-T-C-G), was accomplished by the condensation of the previously described protected nonanucleotide, d[(MeOTr)ibG-anC-T-anC-anC-anC-T-T-bzA], with the trinucleotide block d[pT-anC-mbG(Ac)]. Synthesis of the other segments involved stepwise condensations to the 3'-OH group of growing oligonucleotide chains, starting with suitably protected deoxyribonucleosides and using protected mono-, di-, and trinucleotides as the incoming blocks. The final products, after deprotection, were purified by anion exchange chromatography and characterized. The synthesis of this part of the DNA was planned so that only a hexanucleotide segment is used to go up to the 3'-end (nucleotide 126) of the DNA and, therefore, it is amenable to elongation by chemical methods when the nucleotide sequence of the several nucleotides beyond this end becomes known.

The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.

The DNA sequences of two segments of the ribosomal RNA transcription units of Euglena gracilis Pringsheim strain Z chloroplast DNA have been determined. The first is from the 16 S to 23 S rRNA spacer region. The nucleotide sequence determined includes 64 bp from the 3'-end of the 16 S rRNA gene, the adjacent 87-bp spacer containing 68A-T base pairs, a tRNAIle gene, a 9-bp spacer, a tRNAAla gene, a spacer of approximately 15 bp, and the first 120 bp from the 5'-end of the 23 S rRNA gene. The gene organization of the 16 S to 23 S rRNA spacer, the identity of the tRNA genes, and the tRNA anticodons for the E. gracilis rRNA transcription units are identical with that of the rrnA, D, and X operons of Escherichia coli. The second DNA segment which was sequenced is from a region preceding the 5'-end of the 16 S rRNA gene. Within a continuous region of 189 bp in this 16 S rRNA leader sequence, 68% of the bases are homologous to the 16 S rRNA to 23 S rRNA spacer region. This homology includes the 3'-end of the 16 S rRNA gene, the adjacent spacer, and a complete "pseudo" tRNAIle gene. This leader sequence which has the same polarity as the rRNA transcripts, is flanked by nucleotide sequences resembling partial tRNA genes.

The chemical syntheses of the dodecanucleotide, d(G-C-C-G-C-T-C-G-G-G-A-A), the decanucleotides, d(T-T-T-A-G-A-G-T-C-T), d(G-C-T-C-C-C-T-T-T-G), d(C-G-G-C-C-A-A-A-G-G), and the nonanucleotide, d(G-A-G-C-A-G-A-C-T), are described. The deoxypolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 47-78 of the gene for the tyrosine suppressor tRNA. Chemical syntheses used protected mono- and oligonucleotides and stepwise condensation methods. The detailed plans used are given in Diagrams 1 to 4 in the text. Two additional notable features in the present syntheses were: (a) solvent extraction procedures for the preparation of oligonucleotides as large as pentanucleotides and (b) the demonstration that a heptanucleotide containing 5'-phosphate group can be used successfully in condensation with the 3'-OH group of another oligonucleotide. The synthetic polynucleotides were purified and characterized at protected and unprotected levels.

When V79 pur 1, a purine-requiring auxotroph of a Chinese hamster cell line, is deprived of adenine, nucleic acid and protein synthesis decline rapidly. However, on continuous starvation RNA and DNA synthesis recommences to reach approximately 30% of the normal level between 12 to 24 h starvation. This is accompanied by a rise in the intracellular nucleotide pool. Utilizing mengovirus, which gives a productive infection in V79 pur 1 cells even under conditions of starvation, we can show that rRNA is preferentially degraded and provides the nucleotides for RNA synthesis. Thus "purineless" death in mammalian cells is accompanied by turnover of stable RNA.

The relationship between the methylation and processing of tRNA in both bacterial and mammalian cell systems was investigated by assessing the methylation of an existing population of precursor-tRNAs in the absence of tRNA synthesis. When the synthesis of tRNA in Escherichia coli B (rifampicin) and human KB cells (actinomycin D) was inhibited with the appropriate antibiotic, the incorporation of [3H[methyl groups into tRNA (via [methyl-3H]methionine labeling) rapidly declined with time and was essentially complete within 30 and 60 min, respectively. Although antibiotic treatment predictably reduced the incorporation of methyl groups into tRNA, it also resulted in significant changes in the distribution of the type of methylated products formed. Thus, for KB cells the marked increases in the per cent of radioactivity incorporated into 2'-0-methylribose derivatives, N2-methylguanine, and 3-methylcytosine of tRNA preparation pre-chased with actinomycin D for progressively longer periods of time prior to labeling with [methyl-3H]methionine led to the interpretation that these methylated constituents were formed predominantly during the late stages of tRNA maturation. Similarly, progessive and marked decreases in 1-, 7-, and N2, N2-methylguanine, and moderate decreases in 1-methyladenosine, 5-methylcytosine, and 5-methyluracil revealed that these methylated products were formed primarily during the early and intermediate stages of maturation, respectively. Similar analysis of E. coli B methylation products indicated that the bulk of methyl groups incorporated into the base moieties of tRNA (1- and 7-methylguanine, 2- and N6-methyladenine, and 5-methyluracil) occurred prior to the formation of 2'-0-methylribose derivatives. Additional evidence is presented which negates the possibility that an ancillary action of these antibiotics was the inhibition of specific tRNA-methyl-transferase enzymes.

The total synthesis of a 207-base pair-long DNA, which is biologically functional as a tyrosine suppressor transfer RNA gene, has been completed. The synthesis involved the enzymatic joining of the previously synthesized duplexes. Thus, the duplex corresponding to the promoter region [P] (Sekiya, T., Brown, E.L., Ramamoorthy, B., Fritz, H.-J., Gait, M.J., Lees, R.G., Ryan, M.J., Khorana, H.G., and Norris, K.E. (1979) J. Biol. Chem. 254, 5781-5786) was jointed to Duplex [I] (Caruthers, M.H., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol Chem. 251, 658-666) to form [P + I]. Separatively, Duplex [III + IV + Vb] was prepared from the previously described Duplexes [III], [IV], and [Vb]. (Loewen, P.C., Miller, R.C., Panet, A., Sekiya, T., and Khorana, H.G. (1976) J. Biol. Chem. 251, 642-650; Sekiya, T., Besmer, P., Takeya, T., and Khorana, H.G. 1976) J. Biol. Chem. 251, 634-641; Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G., (1976) J. Biol Chem. 251, 676-694). The product [P + I], was joined to Duplex [II] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol. Chem. 251, 651-657) and then to [III + IV + Vb] without isolation of the intermediates. In all the above joinings, the duplexes carried 32P-labeled phosphate groups at the appropriate 5'-ends. The total DNA and the intermediate duplexes were all characterized by their relative mobilities in electrophoresis on polyacrylamide gel slabs, by nearest neighbor analysis, and by degradation to 5'-nucleotides of radioactively labeled joined products. Two succeeding papers describe the transcription in vitro and the suppressor activity in vivo, of the synthetic gene now described.

The organization of the genes in the rRNA coding region of Euglene gracilis Pringsheim strain Z chloroplast DNA has been determined. 125I labeled 16 S rRNA, 125I labeled 23 S rRNA, and [32P]tRNAs were hybridized to restriction nuclease fragments from cloned chloroplast DNA segments. The coding regions for 16 S and 23 S rRNAs have been located to within 1.75 and 3.1 kbp, respectively. Transfer rRNA genes are located in the 16 S to 23 S rRNA spacer region. Ribosomal RNAs and tRNAs are encoded on the same DNA strand in the 6.2 kbp tandemly repeated rRNA gene region. The order of genes, which is repeated three times in chloroplast DNA, is 16 S rRNA--tRNAS--23 S rRNA--5 S rRNA. Each set of genes is clustered in a region of 5.0 kbp. The organization and size of the genes is as expected if they are all part of a single transcription unit.

Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.

Chemical syntheses of the four deoxyribodecanucleotides, d(T-C-G-A-A-G-T-C-G-A), d(C-G-T-C-A-T-C-G-A-C), d(T-G-A-C-G-G-C-A-G-A), and d(C-T-A-A-A-T-C-T-G-C) are described. These polynucleotides form, respectively, segments 7 to 10 in the plan adopted for the total synthesis of the DNA corresponding to the precursor for the Escherichia coli tyrosine tRNA. The syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. Detailed schemes used in the present syntheses are shown in Diagrams 1 to 4 in the text. The final products were subjected to extensive chromatography and were characterized as pure by chemical and enzymatic procedures.

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.

Two cytoplasmic "petite" (rho-) clones of Saccharomyces cerevisiae have been selected for the retention of the aspartic acid tRNA gene. The two clones, designated DS200/A102 and DS200/A5, have tandemly repeated segments of mitochondrial DNA (mtDNA) with unit lengths of 1,000 and 6,400 base pairs, respectively. The DS200/A102 genome has a single tRNA gene with a 3'-CUG-5' anticodon capable of recognizing the 5'-GAC-3' and 5'-GAU-3' codons for aspartic acid. The mtDNA segment of DS200/A102 has been determined to represent the wild type sequence from 5.3 to 6.8 map units. The genome of DS200/A5 is more complex encompassing the region of wild type mtDNA from 3.5 to 12.7 units. A continuous sequence has been obtained from 3.5 to 8.6 units. In addition to the aspartic acid tRNA, this region codes for the tRNAUGCAla,tRNAUCUArg, tRNAACGArg, tRNAGCUSer,tRNAUCCGly and tRNAUUULys. The DNA sequence of the DS200/A5 genome has allowed us to deduce the secondary structures of the seven tRNAs and to assign precise map positions for their genes. All the tRNAs except tRNA GUCAsp exhibit most of the invariant features of prokaryotic and eukaryotic tRNAs. The aspartic acid tRNA has unusual D and T psi C loops. The structure of this tRNA is similar to the mitochondrial initiator tRNA of Neurospora crassa (Heckman, J.E., Hecker, L.I., Shwartzbach, S.D., Barnett, W.E., Baumstark, B., and RajBhandary, U.L. Cell 13, 83-95).

Chemical syntheses of the pentadecanucleotide, d(G-G-T-G-G-G-G-T-T-C-C-C-G-A-G), the undecanucleotides, d(G-G-T-G-G-G-G-T-T-C-C) and d(C-C-C-C-A-C-C-A-C-G-G), the decanucleotide, d(G-T-A-A-T-G-C-T-T-T), and the nonanucleotides, d(A-T-T-A-C-C-C-G-T) and d(A-G-T-A-A-A-A-G-C) are described. The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 71-103 (from the 3'-end) of the gene for the tyrosine suppressor tRNA. Synthesis of the guanine-rich undecanucleotide d(G-G-T-G-G-G-G-T-T-C-C) was performed by the use of a new protecting group for the guanine ring, the methylbutyryl group. The heptanucleotide d[(MeOTr)mbG-mbG-T-mbG-mbG-mbG-mbG], prepared by the new method, was condensed with the tetranucleotide d[panC-anC-T-T(Ac)]. All of the condensations described followed previously developed chemical principles and started with the N- and 5'-protected deoxyribonucleosides. Successive condensations at the 3'-end with protected mononucleotides, preformed di-, tri-, or tetranucleotides gave products which were separated by anion exchange chromatography and characterized by chemical and enzymatic methods.

Previous studies from this laboratory have provided a high resolution map for 16 tRNA genes located on the continuous heavy DNA strand of bacteriophage T5 DNA (Chen, M.-J., Locker, J., and Weiss, S.B. (1976) J. Biol. Chem. 251, 536-547). All of the T5 tRNA genes were located in three clusters within the DNA C segment, except for tRNAArg, which mapped on the left end of the DNA D segment (Desai, S.M., Hunt, C., Locker, J., and Weiss, S.B. (1978) J. Biol. Chem. 253, 6544-6550). In this report, we present evidence for the presence of eight additional T5 tRNA species, five of which are located in two new loci within the DNA C segment. We also describe a two-dimensional gel electrophoresis system for the separation and isolation of T5 tRNA species from crude infected RNA preparations. The gel electrophoresis system separates tRNA isoacceptors specific for different amino acids; evidence is presented that the isoacceptors for isoleucine, histidine, and serine are coded by different T5 genes.

We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.

The distal region of the tRNA1Tyr gene has been sequenced and found to have an unusual structure. It consists of a 178 base pair sequence that is repeated 3.14 times. The first repeat unit commences 19 base pairs before the end of the sequences encoding the mature tRNA, and these 19 base pairs are repeated faithfully at the beginning of each repeat unit. In the last fractional unit the repeated sequence extends only six base pairs beyond this 19 base pair sequence. Sequence information extends for 62 base pairs beyond the 3.14 repeating units, and no resemblance to the repeating sequence, or any other region of the tRNA1Tyr gene, is found. There are only 14 sites at which one of the repeats differs from the others; 11 of these are transitions, and the rest are transversions. The evolutionary implications of the differences are discussed. One of the differences, which occurs in the second repeat unit, corresponds to the location of the in vitro p-dependent transcription termination site. This is discussed along with other implications of the repeated structure.

This paper reports the evidence for the existence of multiple sites of action of aflatoxin B1 in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hours after aflatoxin B1 injection (0.3 mg/100 g body weight), rat hepatic nuclear and nucleolar RNA synthesis, in vitro, were inhibited 70 and 90% respectively. When total nuclear free and engaged RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 micrograms/ml), only alpha-amanitin-sensitive activity was reduced (50 to 70%) by aflatoxin B1. DEAE-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since aflatoxin B1 dramatically inhibited nucleolar RNA synthesis, but had little effect on RNA polymerase I activity per se, it is concluded, therefore, that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, aflatoxin B1 must also cause impairment of the nucleolar DNA template function.

The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.

The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined. This is: (5') ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5'). The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site. Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts. Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.

Human fibroblasts are shown to incorporate [3H]-mevalonolactone into 3H-labeled delta 2-isopentenyl tRNA. This incorporation was observed in cells that were incubated with compactin (ML-236B), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme. A reductase that blocks mevalonate production by cells. When incubated with low concentrations of [3H]mevalonolactone in the presence of compactin and in the absence of exogenous cholesterol, the cells incorporated small amounts of [3H]mevalonolactone into delta 2-isopentenyl tRNA and large amounts into cholesterol. In the presence of low density lipoprotein, which serves as a source of cholesterol, the incorporation of [3H]mevalonolactone into cholesterol was reduced by 90% and the incorporation into delta 2-isopentenyl tRNA was stimulated by 10-fold. Thus, cultured mammalian cells are now known to use mevalonate for synthesis of three nonsterol products, ubiquinone, dolichol, and delta 2-isopentenyl tRNA, as well as for synthesis of cholesterol.

Several transfer RNA precursors which accumulate in a strain of Escherichia coli temperature-sensitive for RNase P have been described. These precursors range from 135 to 690 nucleotides in length. Their tRNA content has been determined by digestion of the precursors to 4 S RNA, followed by Sanger fingerprint analysis of the purified 4 S material. Identification of some of these tRNAs, as well as an estimate of the number of copies of tRNA in each precursor has been achieved. Many of these precursor RNA molecules contain multiple copies of the same tRNA sequence, indicating a tandem arrangement of the corresponding tRNA genes in the E. coli genome.

Two restriction enzyme fragments containing yeast mitochondrial tRNA genes have been characterized by DNA sequence analysis. One of these fragments is 320 base pairs long and contains a tRNA Ser gene. The corresponding tRNA SER was isolated from yeast mitochondria and its nucleotide sequence also was determined. This mitochondrial tRNA is 90 nucleotides in length, has a G + C content of 38%, and has UGA as the anticodon. A portion of a 680-base-pair DNA fragment containing a tRNA Phe gene was also sequenced. The portion of this gene which codes for the mature tRNA is 75 base pairs in length, has a G + C content of 33%, and contains the anticodon GAA. Neither gene contains an intervening sequence or codes for the 3' CCA terminus. Both are surrounded by regions of more than 90% A + T. The significance of these sequences is discussed.

Duplex [I], which represents the nucleotide sequence 1-26 of the double-stranded DNA corresponding to the precursor for a tyrosine suppressor tRNA, has been synthesized by the enzymatic joining of five chemically synthesized deoxyribooligonucleotide segments. The synthesis was accomplished in two different ways. In a one-step synthesis, all of the five segments were used together: segments 2, 3, and 5 carried 5'-33P-labeled phosphate groups while segment 4 carried a 32P-phosphate group. An alternative, two-step method involved the joining of 5'-32P-phosphorylated segment 2 to segment 4 (carrying 5'-OH group or 5'-32P- or 33P-labeled phosphate group) in the presence of segment 3 followed by the joining of [5-32P]segment 5 in a second step. The duplex [I]' (segments 2 to 5) thus obtained was phosphorlated at the 5'-ends with polynucleotide kinase and then joined to segment 1 to give duplex [I] quantitatively. The preparative methods described have the desired flexibility for performing the subsequent operations necessary for the total synthesis of the structural gene for the tyrosine suppressor tRNA precursor.

Permeabilized cells of Escherichia coli were used to examine the effect on RNA synthesis of guanosine 5'-diphosphate-3'-monophosphate (ppGp). Electrophoretic and hybridization analysis of bulk RNA demonstrated that ppGp selectively reduced the accumulation of both ribosomal and transfer RNAs. The experiments further suggested that the observed reduction in stable RNA accumulation resulted from a reduction in stable RNA chain initiation rather than an effect on elongation, processing, or maturation of stable RNA transcripts. In contrast, the expression of the lac Z gene was not affected by ppGp. These results suggest that ppG, a nucleotide produced in E. coli during the stringent response, could play a direct role in the stringent regulation of stable RNA synthesis.

Chemical synthesis of the undecanucleotides d(T-G-G-G-G-G-A-A-G-G-A), d(C-C-C-C-A-C-C-A-C-C-A), and d(T-C-G-A-A-T-C-C-T-T-C), and the nonanucleotides, d(T-T-C-G-A-A-C-C-T) and d(T-T-C-G-A-A-G-G-T) are described. The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 1-31 (from the 3'-end) of the gene for the tyrosine suppressor tRNA. The synthesis, which basically used the previously developed chemical methods, started with 5' and N-protected deoxyribonucleosides. Successive condensations at the 3'-end were performed using suitably protected mononucleotides or preformed di- and trinucleotides. The condensing agents used were mesitylenesulfonyl chloride or triisopropyl benzenesulfonyl chloride. The required condensation products were isolated partly by solvent partition methods or, in the case of longer chains, by anion exchange chromatography. The completely deprotected deoxypolynucleotides were further purified by anion exchange chromatography in the presence of 7 M urea and characterized by chemical and enzymatic methods.

Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside. When [32P]RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained. All precursors were monomeric. The nucleotide sequences of four asparagine tRNA precursors were determined.

Bacteriophage T4 synthesizes proline and serine tRNA species which are derived from a common precursor RNA. The processing of this precursor RNA involves the replacement of a U-A-A terminus in serine tRNA by C-C-A prior to precursor cleavage. In the present work we have examined in detail the cleavage of T4 proline-serine precursor RNA by the previously identified ribonuclease P. Ribonuclease P accurately cleaves precursor RNA terminating in either C-C-A or U-A-A to generate the 5' termini characteristic of both mature tRNA species. These cleavages do not depend solely on the nucleotide sequence of the precursor RNA since isolated oligonucleotides spanning the cleavage sites are not substrates for the enzyme. Two types of experiments show that RNase P kinetically favors precursor RNA ending C-C-A over that ending U-A-A. Isolated preparations of precursor RNA containing the C-C-A sequence were cleaved more rapidly by RNase P than precursor RNA ending U-A-A. In addition, the serine tRNA generated by limited cleavage of a mixed population of precursor RNA ending C-C-A or U-A-A was enriched 3-fold in the C-A-A sequence relative to the starting material. Bacteriophage T4 proline-serine precursor RNA, in contrast to other tRNA precursors, accumulates in measurable amounts in wild type cells. This accumulation would appear to be a consequence of the requirement for the generation of the C-C-A sequence prior to RNase P cleavage. The enzymic specificity of RNase P in vitro therefore reflects the in vivo pathway for serine tRNA biosynthesis, where the C-C-A sequence is synthesized while the serine tRNA sequence is still a part of the large precursor RNA.