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Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions., Competing Interests: The authors declare no conflict of interest.

In eukaryotes, transfer RNAs (tRNAs) are transcribed in the nucleus yet function in the cytoplasm; thus, tRNA movement within the cell was believed to be unidirectional--from the nucleus to the cytoplasm. It is now known that mature tRNAs also move in a retrograde direction from the cytoplasm to the nucleus via retrograde tRNA nuclear import, a process that is conserved from yeast to vertebrates. The biological significance of this tRNA nuclear import is not entirely clear. We hypothesized that retrograde tRNA nuclear import might function in proofreading tRNAs to ensure that only proper tRNAs reside in the cytoplasm and interact with the translational machinery. Here we identify two major types of aberrant tRNAs in yeast: a 5', 3' end-extended, spliced tRNA and hypomodified tRNAs. We show that both types of aberrant tRNAs accumulate in mutant cells that are defective in tRNA nuclear traffic, suggesting that they are normally imported into the nucleus and are repaired or degraded. The retrograde pathway functions in parallel with the cytoplasmic rapid tRNA decay pathway previously demonstrated to monitor tRNA quality, and cells are not viable if they lack both pathways. Our data support the hypothesis that the retrograde process provides a newly discovered level of tRNA quality control as a pathway that monitors both end processing of pre-tRNAs and the modification state of mature tRNAs.

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation.

Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable preinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short ( approximately 100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high reinitiation rate. Transcription reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.

Three NifS-like proteins, IscS, CSD, and CsdB, from Escherichia coli catalyze the removal of sulfur and selenium from L-cysteine and L-selenocysteine, respectively, to form L-alanine. These enzymes are proposed to function as sulfur-delivery proteins for iron-sulfur cluster, thiamin, 4-thiouridine, biotin, and molybdopterin. Recently, it was reported that selenium mobilized from free selenocysteine is incorporated specifically into a selenoprotein and tRNA in vivo, supporting the involvement of the NifS-like proteins in selenium metabolism. We here report evidence that a strain lacking IscS is incapable of synthesizing 5-methylaminomethyl-2-selenouridine and its precursor 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA, suggesting that the sulfur atom released from L-cysteine by the action of IscS is incorporated into mnm(5)s(2)U. In contrast, neither CSD nor CsdB was essential for production of mnm(5)s(2)U and 5-methylaminomethyl-2-selenouridine. The lack of IscS also caused a significant loss of the selenium-containing polypeptide of formate dehydrogenase H. Together, these results suggest a dual function of IscS in sulfur and selenium metabolism.

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

RPP2, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of Saccharomyces cerevisiae. Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P. Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells. Depletion of Rpp2 protein in vivo causes accumulation of precursor tRNAs with unprocessed introns and 5' and 3' termini, and leads to defects in the processing of the 35S precursor rRNA. Rpp2-depleted cells are defective in processing of the 5.8S rRNA. Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor rRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30. These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans.

We have previously described the mutator alleles mutA and mutC, which map at 95 minutes and 42 minutes, respectively, on the Escherichia coli genetic map and which stimulate transversions; the A.T-->T.A and G.C-->T.A substitutions are the most prominent. In this study we show that both mutA and mutC result from changes in the anticodon in one of four copies of the same glycine tRNA, at either the glyV or the glyW locus. This change results in a tRNA that inserts glycine at aspartic acid codons. In view of previous studies of missense suppressor tRNAs, the mistranslation of aspartic acid codons is assumed to occur at approximately 1-2%. We postulate that the mutator tRNA effect is exerted by generating a mutator polymerase and suggest that the epsilon subunit of DNA polymerase, which provides a proofreading function, is the most likely target. The implications of these findings for the contribution of mistranslation to observed spontaneous mutation rates in wild-type strains, as well as other cellular phenomena such as aging, are discussed.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.

Some mitochondrial tRNA genes of land snails show mismatches in the acceptor stems predicted from their gene sequences. The majority of these mismatches fall in regions where the tRNA genes overlap with adjacent downstream genes. We have synthesized cDNA from four circularized tRNAs and determined the sequences of the 5' and 3' parts of their acceptor stems. Three of the four tRNAs differ from their corresponding genes at a total of 13 positions, which all fall in the 3' part of the acceptor stems as well as the discriminator bases. The editing events detected involve changes from cytidine, thymidine, and guanosine to adenosine residues, which generally restore base-pairing in the stems. However, in one case an A-A mismatch is created from an A-C mismatch. It is suggested that this form of RNA editing may involve polyadenylylation of the maturing tRNAs as an intermediate.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.

The function of a TATA element in RNA polymerase (EC 2.7.7.6) III transcription of a naturally TATA-containing U6 snRNA gene and a naturally TATA-less tRNA gene was probed by transcription and Ty3 transposition analyses. Deletion of the TATA box from a U6 minigene did not abolish transcription and Ty3 integration but changed the positions of initiation and insertion. Insertion of the U6 TATA box at three positions upstream of the TATA-less SUP2 tRNA(Tyr) gene resulted in novel transcription initiation and Ty3 integration patterns that depended upon position of the insertion. Nevertheless, the predominant tRNA gene initiation sites were not affected by insertion of the TATA sequence and remained at a fixed distance from the internal box A promoter element. Insertions of the TATA box upstream of a SUP2 box A mutant affected the level of transcription and restricted the use of upstream start sites, but they neither enhanced the use of TATA-dependent initiation sites nor restored expression to the level of the wild-type gene. We conclude that (i) the U6 TATA box is essential in vivo for correct initiation but not for transcription, (ii) a TATA box does not compensate for a weak box A sequence and so cannot perform equivalently, and (iii) the TATA-binding protein, and probably components of transcription factor IIIB, are present on the target at the time of Ty3 integration.

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.

The sequence of 29 nucleotides immediately preceding the starting point of transcription of the E. coli tyrosine tRNA gene has been determined. This is: [Formula: see text] The sequence contains regions of 2-fold symmetry. Transcription of the gene begins at the first nucleotide to the left of nucleotide 1 and is leftward.

Methionine analogs such as ethionine, selenomethionine, and trifluoromethionine all arrest growth and division of the yeast Saccharomyces cerevisiae. One analog, ethionine, caused cells of the yeast to arrest specifically within G1; reciprocal shift experiments showed that ethionine and alpha-factor arrested cells at the same step ("start"). The major effect of ethionine on synthesis of macromolecules was to reduce both the rate of appearance of 35S ribosomal precursor RNA and the rate of production of mature rRNA. Synthesis of protein was relatively unaffected by ethionine. Selenomethionine and trifluoromethionine caused cells to arrest randomly in the cell division cycle. Although treatment of cells with either selenomethionine or trifluoromethionine also reduced the rate of total RNA synthesis, each of these analogs had other effects that presumably prohibited completion of the cell cycle. We propose that the rate of rRNA production is an important regulatory event in the cell cycle.

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.

RNA ligase from T4-phage-infected Escherichia coli cells catalyzes the covalent joining of two polynucleotides that are partially hydrogen-bonded to each other. Two polynucleotide fragments derived from yeast tRNA(Phe) and consisting of residues 1-36 and 38-74 are covalently joined by the enzyme. The product of the reaction lacks residue Y(37) and has an anticodon loop with six nucleotide residues, whereas all tRNA species whose sequences have so far been determined have seven nucleotides in this loop. Evidence is also presented for the formation of a polynucleotide-adenylylate intermediate in the joining reaction, in which a pyrophosphate bond links the 5'-phosphoryl terminus of the polynucleotide and the phosphoryl group of AMP.

Chromatin prepared by gentle methods from mouse myeloma cells retained its ability to synthesize RNA using bound endogenous RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). The transcription resembles that observed in vivo in several respects. The low-molecular-weight RNA species 5S RNA and the 4.5S precursor to 4S RNA, are transcribed accurately and transcription is reinitiated continually in vitro. Their synthesis was not inhibited by alpha-amanitin (1 mug/ml) as was found previously for these species in isolated nuclei.

Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated III(A) and III(B)). The enzymes are unaffected by low alpha-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for III(A) and III(B)) at higher toxin concentrations. RNA polymerase I was unaffected at all alpha-amanitin concentrations tested. Myeloma RNA polymerases II, III(A), and III(B) appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic alpha-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases. Isolated myeloma nuclei and nucleoli contińue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by alpha-amanitin, but only at high concentrations; and the alpha-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases III(A) and III(B). In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by alpha-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high alpha-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems. These results unequivocally demonstrate the role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species.

RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates.

The nucleotide sequence of a transfer RNA precursor molecule coded by bacteriophage T4 has been determined. The molecule is a single polynucleotide chain which contains two transfer RNA species that are destined to recognize serine and proline. The 3' -CCA(OH) termini of both mature transfer RNA species are absent in the precursor molecule; these termini must therefore be added enzymatically at a subsequent stage of maturation. Nucleotide residues unique to the precursor are located at both ends of the molecule and between the two transfer RNA sequences.

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.

Cloned tRNA genes from Drosophila and from yeast have been transcribed faithfully in extracts prepared from Xenopus germinal vesicles. The newly formed RNA is composed of precursor tRNAs (of 5S RNA size) and of tRNAs. The plasmid pCIT12 carries genes for Drosophila tRNALys, tRNAArg, and tRNAAsn, Nucleotide analysis of one RNA species transcribed from pCIT12 DNA showed it to be identical to Drosophila tRNALys; it even contained some of the modified nucleotides expected for this tRNA. This RNA species is formed in the germinal vesicle extract via a larger precursor tRNA molecule that does not contain nucleotide modifications. This simple transcription system should aid studies aimed at defining the regulatory DNA regions responsible for eukaryotic gene transcription. In addition, it may provide tRNA precursors that are needed for detailed investigations of eukaryotic tRNA biosynthesis.

Certain nucleotides in precursor RNA of tRNA(Tyr) of Escherichia coli were modified in vitro with a preparation of partially purified E. coli enzyme containing ribothymidine- and pseudouridine-forming activity. The only nucleotides modified in vitro are the same as those found modified in mature tRNA. The best substrate for these modifying enzymes is the RNase P cleavage product of the precursor RNA, which contains the mature tRNA sequence. Of the two pseudouridines found in mature tRNA, one (in the TPsiC sequence) can be formed in intact precursor RNA. The other (in the anticodon stem) can only be formed in the cleaved precursor RNA. The presence of modified nucleotides in the precursor RNA does not enhance its rate of cleavage by RNase P.

Ribosylthymine is not present in tRNA of Streptococcus faecalis grown in the absence of folic acid, although this methylated residue does occur in the tRNA of this organism when it is grown in the presence of folate. We have found that, unlike other methylated residues in RNA of S. faecalis and other organisms, the methyl moiety of ribosylthymine of the tRNA of folate-sufficient S. faecalis is not derived from S-adenosylmethionine. Preliminary evidence suggests that a folate derivative serves as the methyl donor in this methylation.

Successive rounds of mutagenesis of a Schizosaccharomyces pombe strain bearing the UGA-reading sup3 tRNASer suppressor have been carried out for two cycles of inactivation and reactivation of the suppressor. The suppressor phenotype at each stage was found to involve different combinations of three mutations, A30, A53, and A67, in the sup3-UGA gene. Single mutations A30 and A53 inactivate the suppressor as does the presence of all three mutations. A67 by itself is phenotypically neutral, but in combination with either A30 or A53 suppressor function is restored. The frequency with which these and other complementation events occur in S. pombe demonstrates a significant potential for nucleotide sequence evolution in tRNA. Differential expression of the S. pombe genes in Saccharomyces cerevisiae suggests that the two yeasts have diverged at the transcriptional and RNA processing level. Processing of the mutant tRNA precursors in S. cerevisiae reveals a hierarchy of structural domains within the tRNA that vary in their importance for RNase P cleavage.

A Xenopus laevis DNA segment containing the structural gene for tRNA1Met and 22 base pairs at the 5' side of the gene is active in tRNA production. The DNA segment was ligated to sea urchin histone DNA and was also inserted into plasmid pCR1. Both recombinant DNAs were shown to produce mature tRNA1Met at a high rate when injected into centrifuged Xenopus oocytes.

By RNA-DNA hybridization, as well as chemical and chromatographic analysis, evidence is provided that the bacteriophage T5 codes for the synthesis of two isoacceptor methionine transfer RNA species, tRNA-Met and tRNA-f-Met. Because of the differences in chromatographic properties of T5 phage and host methionine tRNAs, the phage tRNA species are readily distinguishable.

The three major RNA classes from zinc-sufficient [(+Zn)] and zinc-deficient [(=Zn)] Euglena gracilis have been separated by affinity chromatography on oligo(dT)- and N-[N'-[m-(dihydroxyboryl)phenyl]succinamoyl]aminoethyl (DBAE)-celluloses. The total RNA content and the ribosomal and transfer RNA fractions are the same in (+Zn) and (=Zn) cells. IN (-Zn) cells, the messenger RNA fraction increases, and its altered base composition reveals additional bases and a 2-fold increase in the (G+C)/(A+U) ratio. Since the intracellular content of manganese increases in (-Zn) cells, we have examined its role in determining these changes in RNA composition. An increase in the Mn2+ content from 1 to 10 mM in assays with RNA polymerases I and II from (+Zn) cells and those with the single RNA polymerase from (-Zn) cells decreases the ratio of UMP to CMP incorporated from 1.7 to 1.0, 2.1 to 0.8 and 3.5 to 0.4, respectively. Thus, Mn2+ concentration can significantly alter the products of the enzymatic action of RNA polymerases from both (+Zn) and (-Zn) E. gracilis cells.

In a temperature-sensitive mutant of E. coli defective in tRNA biosynthesis, many tRNA precursors, including monomeric and multimeric forms, accumulate. Some of the multimeric precursors contain three or more tRNA sequences within a molecule. These large precursors were cleaved by cell extracts first into intermediate size pieces which were subsequently processed by RNase P. On the basis of heat stability of mutant cell extracts, the endonuclease responsible for the initial cleavage appears to be distinct from RNase P and is designated RNase O. One of the monomeric precursors was shown to be processed first by RNase P and the product subsequently cleaved further into a smaller molecule. The nuclease responsible for this second cleavage also appears to be distinct from RNase P and is designated RNase Q. The functions of these nucleases are sequential in the trimming process with respect to that of RNase P; RNase O works prior to RNase P and RNase Q after RNase P but in both cases, not vice versa.

All sequenced histidine tRNAs have one additional nucleotide at the 5' end when compared to other tRNA species. Sequence analysis of histidine tRNA genes from Drosophila melanogaster and Schizosaccharomyces pombe showed that the terminal guanylate residue of the mature tRNAs is not encoded by the genes. Analysis of the products from in vitro transcription of these genes in extracts from Drosophila Kc cells demonstrated that the 5'-terminal nucleotide present in the mature tRNA is added post-transcriptionally. The addition reaction requires ATP. A portion of the mature tRNAs are then modified at the 5'-terminal pG. Analysis of the RNA species formed during the in vitro maturation of the Drosophila histidine tRNA primary transcript uncovered the following maturation scheme: (i) the primary transcript is processed by RNase P at the 5' end to form an intermediate precursor; (ii) the 3'-flanking sequence is endonucleolytically removed, and a guanylate moiety is added to the 5' end to form mature-sized histidine tRNA; and (iii) a fraction of the 5'-terminal guanylate residues then undergoes modification. In contrast to the capping of eukaryotic mRNA, the guanylate addition to histidine tRNA results in the formation of a (3'-5')-phosphodiester bond. There are no precedents for the post-transcriptional addition of nucleotides (in phosphodiester linkage) to the 5' end of RNA precursors.

We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans.

The hisT gene codes for an enzyme responsible for the conversion of uridine to pseudouridine (Psi) in the anticodon region of many tRNA species in Salmonella typhimurium. We have previously shown that a hisT mutant has tRNA(His) which lacks pseudouridine in this region and as a consequence has an altered chromatographic behavior. We show here a similar alteration in chromatographic behavior of all tRNA(Leu) and one tRNA(Ile) species from a hisT mutant. By contrast, tRNA(Val), which contains no pseudouridine except for the one in the TPsiCG sequence, is chromatographically unaltered in a hisT mutant. The absence of pseudouridine in the anticodon region of tRNA in hisT mutants has been previously shown to cause derepression of the histidine operon. We show here that in hisT mutants the regulation of the leucine and the isoleucine and valine operons is also affected: the enzymes of these operons are refractory to repression by the branched chain amino acids. However, there is no difference between hisT and wild type in the pattern of derepression caused by isoleucine or valine limitation and only a slight difference in the enzyme levels in cells grown on minimal medium. The alteration in the regulation of branched chain amino acid operons may also explain why hisT mutants are resistant to inhibition of growth by the amino acid analogues 5,5,5-trifluoroleucine, beta-hydroxyleucine, and norleucine and by the oligopeptides glycylglycylnorleucine and norleucylnorleucine.

By using sites for the restriction nuclease Hpa II, the information for the anticodon stem and loop of an altered Su+2 amber suppressor tRNA (a mutant of tRNAGln) has been transplanted to a specially prepared tRNATrp gene, which lacks it homologous anticodon stem and loop sequence. The resulting tRNA gene was cloned under lac operator-promoter control. The result is a functional, hybrid, amber-suppressor tRNA that can exhibit a moderately high efficiency in translation. It appears less efficient, however, than Su+7 tRNA, the amber suppressor that results from a direct anticodon mutation in tRNATrp. As judged by its suppressor spectrum, which is almost identical to the spectra of Su+2, and Su+7, the recomposed tRNA inserts glutamine at amber sites. This experiment is the prototype of a series of construction that examine the role of the nucleotides in the anticodon region.

Bacteriophage T4 serine and proline transfer RNAs are derived from a common precursor RNA. This precusor RNA lacks -C-C-A sequences which could provide 3' termini for the mature transfer RNAs. We have deduced part of the pathway leading to the formation of the C-C-A sequences in the transfer RNAs by characterizing incompletely matured precursor molecules which accumulate during infection of mutant hosts that lack specific enzymes associated with transfer RNA metabolism. Maturation is initiated by the addition of -C-C-AOH to the 3' terminus of the precusor RNA through the combined actionof an unidentified nuclease and tRNA nucleotidyltransferase (EC 2.7.7.25). Precursor RNA molecules terminating in -C-C-AOH is serine transfer RNA and the second product is immature proline transfer RNA. The terminal steps leading to proline transfer RNA have not been fully delineated, but are known to involve the replacement of a -C-UOH sequence by -C-C-AOH.

Short-labeled 4.5S RNA molecules isolated from the posterior silk gland of Bombyx mori can be separated by two-dimensional polyacrylamide gel electrophoresis into many discrete species, some of which are radiochemically pure by the criteria of RNA fingerprinting. One region of the gel contains two precursor RNAs, one to each of the known alanine transfer RNAs. Each precursor tRNAAla molecule contains all of the internal oligonucleotides present in the corresponding tRNAAla species plus new 5'-and 3'-terminal sequences. Precursor molecules to tRNA1Gly (which differ from each other in size) are contained in two other gel regions, and a fourth region contains a precursor to tRNA2Gly. Both of the transcription initiator purine tetraphosphate nucleosides are present in unfractionated tRNA precursor mixtures, with pppA- predominating over pppG-. Minor nucleotides are also present in B. mori tRNA precursors. No polycistronic tRNA gene transcripts were observed.

Evidence for the presence of rapidly labeled short-lived RNA species, presumed to be precursors to transfer RNA (tRNA), in the silk glands of Bombyx mori is presented. These precursors, which migrate between 4S and 5S markers on acrylamide gels, can be converted in vitro into molecules indistinguishable in size from tRNA. The conversion is also catalyzed by a crude enzyme fraction isolated from ribosomes of E. coli. Since a similar enzyme preparation cleaves the suppressor tRNA(Tyr) sequence of E. coli from its precursor molecule, it would appear that the precursor to E. coli tRNA(Tyr) and the precursors to silk-gland tRNAs share common structural features.

The formation of trifluoroleucine-tRNA and leucine-tRNA during embryonic development of the mouse is a function of gestational age. The existence of a leucine-tRNA synthetase has been demonstrated in early embryonic tissue (gestational age <10 days) which has unique chemical properties, acylating tRNA with trifluoroleucine with avidity almost equal to leucine. This enzyme has been shown to be stable to heating at 45 degrees C for 30 minutes.A second leucine-tRNA synthetase, which is similar chemically and physically to the enzyme isolated from more mature embryonic tissue, has been isolated from this embyronic source as well as from adult tissue. Quantitative changes in the species of tRNA isolated from these various sources have been documented utilizing the technique of polyacrylamide electrophoresis. These changes parallel the disappearance of the early embryonic enzyme, fewer species of tRNA acting as acceptors for leucine as maturation progresses.

Biologically active su(+) (III) tyrosyl-tRNA has been synthesized in a DNA-directed cell-free system from Escerichia coli. Such a system should be most useful for studying the mechanism of tRNA synthesis. This tRNA is capable of suppressing amber mutations in the gene coding for beta-galactosidase (EC 3.2.1.23) and, therefore, must be capable of being charged and transferring amino acids. A 4-fold stimulation in the activity of the tRNA formed de novo is obtained with isopentenyl pyrophosphate, a compound involved in the post-transcriptional acylation of an adenine base adjacent to the anticodon. It has been suggested elsewhere that formation of RNA subject to stringent control may be inhibited by guanosine tetraphosphate (ppGpp). However, guanosine tetraphosphate did not affect the synthesis of su(+)III tyrosyl-tRNA, even though the synthesis of this tRNA is subject to stringent control.

A defective varphi80 transducing phage has been isolated that carries a small section of the E. coli chromosome from the region near argH (77 min). This phage contains three E. coli tRNA genes, termed glyTsu(+) (36), tyrT, and thrT. These genes specify the structures of tRNA(2) (Gly) (su(+) (36)) tRNA(2) (Tyr), and tRNA(3) (Thr), respectively. Induction of a single lysogen bearing this phage brings about selective synthesis and amplification of only these three tRNAs.

During infection of Escherichia coli, bacteriophage T4 directs the synthesis of at least eight transfer RNAs and of two stable RNA species of low molecular weight, of unknown function. When T4 DNA is incubated with purified RNA polymerase and the appropriate substrates, a high molecular weight RNA is produced. This RNA, on further incubation with a supernatant fraction prepared from E. coli, is cleaved to several species of RNA. These cleaved RNAs were analyzed by gel electrophoresis and fingerprint techniques, and were found to be similar or identical to those made in vivo. The fingerprint analysis of one of these, a tRNA(Gly), is presented. The molecule made in vitro, except for the absence of modified bases, appears to be identical to the RNA made by T4-infected cells. Therefore, in this system the tRNA genes are transcribed with fidelity, the transcript is cleaved correctly, and the tRNAs are made in good yield.

The binding of phenylalanyl-oligonucleotide, C-A-C-C-A-(Phe), to ribosomes requires the presence of a free amino group on the amino acid. Acetylation of the amino acid reduces the binding to ribosomes to about 1/20 of the binding observed with the intact Phe-oligonucleotide. Deacylation of the phenylalanyl-oligonucleotide eliminates binding of the free amino acid and markedly reduces the binding of the oligonucleotide (C-A-C-C-A). Neither 30S nor 50S subunits alone are sufficient for binding of the phenylalanyl-oligonucleotide; the presence of both subunits is necessary. The data suggest that phenylalanyl-oligonucleotide binding to ribosomes represents the binding of the aminoacyl-terminus of phenylalanyl-tRNA and that the presence of an amino acid with an unsubstituted amino group attached to the oligonucleotide (C-A-C-C-A) is required for binding to this potassium-dependent site. Furthermore, the ribosome itself has the capability of distinguishing between aminoacyl-oligonucleotides with N-substituted and unsubstituted amino acids-

Bacteriophage T4 induces the synthesis of eight transfer RNAs upon infection of E. coli. The tRNAs are easily detected and resolved into pure species by polyacrylamide gel electrophoresis of RNA labeled with (32)P after T4 infection. Two-dimensional fingerprints of RNase T(1) products derived from individual gel bands give patterns characteristic of single tRNAs. Furthermore, the T(1) digest of each gel band has a single oligonucleotide that contains the minor nucleotides Tp and Psip, a characteristic feature of all known tRNAs. Four larger RNAs are also seen in the polyacrylamide gels. Fingerprint and genetic analyses demonstrate that these molecules are related to the tRNAs, but the exact nature of this relationship is not known.

All types of RNA are actively synthesized by the lethal hybrid obtained by fertilizing the eggs of the sea urchin Paracentrotus lividus with the sperm of another sea urchin species, Arbacia lixula. DNA-like RNA and ribosomal RNA seem to be less stable in hybrids than in normal A. lixula and P. lividus embryos. Labeled RNA's from A. lixula and P. lividus were found to hybridize about 2.5 times as well with DNA of their own species as with DNA of the other species. RNA from the interspecific hybrid hybridized from 1.6 times to twice as well with A. lixula DNA as with P. lividus DNA, although the hybrid embryos were previously shown(5) to contain more DNA of maternal than of paternal type. Transcription of the paternal (foreign) genome seems therefore to be favored in some way in the interspecific hybrid.

Using a simple three-step procedure, we have isolated thermosensitive mutants of E. coli that are specifically defective in transfer RNA (tRNA) synthesis. Our procedure was designed to identify mutants that are unable to make su(3) (+) tRNA(Tyr) or grow at the restrictive temperature, yet under the same conditions they retain the ability to make mRNA and protein. The mutants obtained have been analyzed, and they are defective in different steps in the synthesis of functional tRNA at the restrictive temperature. Some of them may fail to modify certain bases in the tRNA. Two mutants are unable to process the 5' end of tRNA precursor molecules. Three are unable to cleave precursor molecules at the 3' end. 11 Mutants can not synthesize any tRNA molecules or tRNA precursors. We speculate that these latter mutants may be defective in RNA polymerase or in an RNA polymerase factor specific for stable RNA synthesis.