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Cytoplasmic rRNA from the yeast mutant, mod5-1 is deficient in the modified base isopentenyladenosine and consequently migrates differently from isopentenylated wild type tRNAs on certain chromatographic systems. To determine if the mod5-1 mutation affects mitochondrial tRNA structure, the migration of mitochondrial tRNA from mod5-1 and wild type cells has been compared by reverse-phase chromatography. We conclude from this analysis that the single nuclear mutation that affects the isopentenylation of cytoplasmic tRNA also affects the isopentenylation of mitochondrial tRNA.

Yeast mitochondrial DNA-pBR322 recombinant DNA molecules known to contain tRNA genes from a tRNA rich region of the yeast genome were used as a source of DNA for restriction mapping and tRNA gene sequence analysis. We report here restriction maps of two segments of yeast mitochondrial DNA and the sequence of mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR. Both genes are flanked by A + T rich DNA and neither has an intervening sequence nor codes for a 3' CCA end. The tRNA structures deduced from the genes have the usual cloverleaf structures and invariant nucleotides. This combination of DNA sequencing and restriction mapping has enabled us to determine that the tRNAvalGUR and a previously sequenced tRNA, the tRNApheUUY are transcribed from the same strand of DNA.

A number of novel observations on ribosomal metabolism were made during gametic differentiation of Chlamydomonas reinhardi . Throughout the gametogenic process the amount of chloroplast and cytoplasmic ribosomes decreased steadily. The kinetics and extent of such decreases were different for each of the two ribosomal species. Comparable rRNA degradation accompanied this ribosome degradation. Concurrent with the substantial ribosome degradation was the synthesis of rRNA, ribosomal proteins and the assembly of new chloroplast and cytoplasmic ribosomes throughout gametogenesis. The newly synthesized chloroplast ribosomes exhibited distinctively faster turnover than their cytoplasmic counterpart. Cytoplasmic ribosomes, pulse-labeled in early gametogenic stages, retained label until differentiation was nearly complete even though a net decrease in the level of cytoplasmic ribosomes continued, indicating that the newly synthesized cytoplasmic ribosomes were preferentially retained during differentiation. Hence the regulation of ribosome metabolism during gametogenesis contrasts with the conservation of ribosomes obtained during vegetative growth of C. reinhardi and other organisms. This unique pattern of ribosome metabolism suggests that new ribosome synthesis is necessary during gametogenesis and that some specific structural or functional difference relating to the development stage of the life cycle might exist between degraded and newly synthesized ribosomes.

Yeast mitochondrial DNA-pBR322 recombinant DNA molecules screened for tRNA genes were used as a source of DNA for mitochondrial tRNA gene sequence analysis. We report here the sequences of yeast mitochondrial tRNA genes coding for a tRNA AGY Ser and a tRNA CGY Arg . The tRNA AGY Ser sequence deduced from the gene is the first reported sequence of a yeast tRNA AGY Ser . It is also the second yeast mitochondrial tRNA Ser gene to be sequenced, and demonstrates unequivocally the presence of mitochondrial encoded serine tRNA isoacceptors. The tRNA CGY Arg sequence deduced from the gene is the most AT-rich (82%) tRNA sequence ever reported. Whereas all the mitochondrial genes sequenced to date exist singly on the genome and are separated by long stretches of AT-rich DNA, the tRNA AGY Ser and tRNA CGY Arg genes exist in tandem, separated by only 3 bp. This gene arrangement strongly suggests that mitochondrial tRNA genes may be transcribed into multicistronic precursors.

The effects of phenobarbital (PB)-delayed ovulation on embryonic development, implantation, and serum concentrations of progesterone (P), androstenedione (A), and estradiol (E2) were ascertained in the pregnant mare serum gonadotropin (PMSG)-treated immature rat. Two and three days of ovulatory delay retarded embryonic development as evidenced by the presence of 70-80% of the embryos in the two-cell stage on Day 3 (Day 1 = sperm positive); whereas, nondelayed controls exhibited 40-50% of the embryos in the two-cell stage and 30-40% in the four-cell stage. Ovulatory delay of 3 days induced a higher percentage of abnormal embryos when compared with controls. After 2 days of delay, implantation was inhibited in ~40% of the rats by Days 6 and 8. In controls ~80% of rats exhibited implantation sites on Day 6 and all control rats exhibited implantation sites on Day 8. Serum concentrations of P, A, and E2 were similar to nondelayed controls on Day 3 of pregnancy suggesting that the retardation in develop...

A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.

We have previously described a mitochondrial activity that removes 5' leaders from yeast mitochondrial precursor tRNAs and suggested that it is a mitochondrial RNase P. Here we demonstrate that the cleavage reaction results in a 5' phosphate on the tRNA product and thus the activity is analogous to that of other RNase Ps. A mitochondrial gene called the tRNA synthesis locus encodes an A + U-rich RNA required for this activity in vivo. Two regions of this RNA display sequence similarity to conserved sequences in bacterial RNase P RNAs. This sequence similarity coupled with the analogous activities of the enzymes has led us to conclude that the RNAs are homologous and that the tRNA synthesis locus does code for the mitochondrial RNase P RNA subunit. The smallest and most abundant transcript of the tRNA synthesis locus is 490 nucleotides long. However, during purification of the holoenzyme, RNA is degraded and pieces of the original RNA are sufficient to support RNase P activity in vitro.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs. By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene. The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp. The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp. The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end. We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts. The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

To delineate the total number of tRNAs encoded by yeast mitochondrial DNA (mtDNA), we have examined mitochondrial tRNA preparations for the presence of heterogenic isoaccepting tRNAs. Analyses of /sup 3/H-labeled aminoacylated mitochondrial tRNAs by reversed-phase column chromatography (RPC-5) coupled with hybridization to mtDNA detected only one major mitochondrially coded tRNA for alanine, arginine, aspartic acid, glycine, histidine, isoleucine, leucine, lysine, proline, serine, and tryptophan. Some of these profiles also contained one or more minor peaks that may represent small amounts of heterogenic isoacceptors, but their low concentrations prevented their characterization. Cysteinyl-, methionyl-, phenylalanyl-, threonyl-, tyrosyl-, and valyl-tRNAs separated into multiple species upon RPC-5 chromatography. Two cysteinyl-, two methionyl-, two phenylalanyl-, two threonyl-, four tyrosyl-, and two valyl-tRNA species hybridized to mtDNA. The hybridization of the phenylalanyl- and the valyl-tRNA species was not additive, indicating that their sequences are similar, if not identical, and suggesting that they may be transcribed from the same genes. The two methionyl-, and two threonyl-, and probably the two cysteinyl-tRNAs are transcribed from separate genes, since their hybridizations to mtDNA are additive. That the methionyl-tRNAs are transcribed from separate genes was further confirmed through deletion mapping experiments which showed that the genes coding for these tRNAsmore » are at different locations on the mtDNA. The transcriptional relationship of the four tyrosyl-tRNAs was not established. There is at least a cistron coding for tRNAs corresponding to each of the common amion acids except asparagine and several amino acids (methionine, threonine, and cysteine) are accepted by more than one transcriptionally distinct tRNA. Thus, a minimum of 22 tRNA cistrons have been identified.« less

The trm1 mutation of Saccharomyces cerevisiae is a single nuclear mutation that affects a specific base modification of both cytoplasmic and mitochondrial tRNA. Transfer RNA isolated from trm1 cells lacks the modified base N2,N2-dimethylguanosine, and extracts from these cells do not have detectable N2,N2-dimethylguanosine-specific tRNA methyltransferase activity. As part of our efforts to determine how this mutation affects enzyme activities in two different cellular compartments we have isolated the TRM1 locus by genetic complementation. The TRM1 locus restores the N2,N2-dimethylguanosine modification to both cytoplasmic and mitochondrial tRNA in trm1 cells. An open reading frame in this TRM1 gene is essential for complementation of the trm1 phenotype. Expression of this open reading frame in Escherichia coli converts the organism from one that neither makes N2,N2-dimethylguanosine nor has N2,N2-dimethylguanosine-specific tRNA methyltransferase activity into one that does. This result suggests that the TRM1 locus is the structural gene for the tRNA modification enzyme and that both nuclear/cytoplasmic and mitochondrial forms of the methyltransferase are produced from the same gene.

Phenobarbital blockade of ovulation for 3 days in the proestroushamster induces atresia of preovulatory follicles. In vitro incubations of the delayed preovulatory follicles with luteinizing hormone (LH) revealed that as the follicles approach the atretic phase (Day 3 ofdelay)theylose the ability to produce androstenedione (A) and estradiol (E2) but acquire the capacity to produce large quantities of progesterone (P) (Terranova, 1981b). This shift in steroidogenesis, from E2, to P, resembles those alterations in steroidogenesis during the preovulatory period in response to the LH/follicle-stimulating hormone (FSH) surge. The aim of the present study was to determine the sourceof P in the delayed follicle by in vitro incubation of theca and granulosa, separately, and to contrastand compare steroidogenesis ofthe delayed follicle with preovulatory follicles before and after the LH/FSH surge inthehamster.IncubationofthecawithLH on proestrusand eachday of delay(Days 1-3) revealedthatthecaisa primary source of follicular A and that 2-3 days of ovulatorydelayinducesa decline in the ability of thecato produce A in vitro. Concomitant with the decline in theca A production in vitro was an increaseinthecal P production. A similar shift from A to P production by theca in vitro was observed in preovulatory follicles exposed to an endogenous LH/FSH surge.Granulosacellsfrom proestrous follicles (before the LH surge) and from delayed follicles did not accumulate P in vitro. However after the LH/FSH surge granulosa cellsfrom nondelayed proestrous follicles produced large amounts of P in vitro.Granulosa cells aromatized more A to E2 on Days 1 and 2 ofdelaythannondelayedcontrols; however,by Day 3 of delayaromatizingactivity was reduced. These results indicate that theca is the major source of P in delayed follicles approaching the atretic phase; whereasin post-LH/FSH surge follicles both theca and granulosa cells produce significant quantities of P. In addition, ovulatory delay induced a significant decline in theca A production in vitro similar to that observed in preovulatory follicles after the LH/FSH surges.

The calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli . Expression of soluble protein was optimal at 15°C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity ( K i 0.3–1 nM) for cathepsin L and papain, and a somewhat lower affinity ( K i ∼ 15 nM ) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated.

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNA f Met gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.

Publisher Summary This chapter deals with Glu-tRNA Gln , which is an intermediate in yeast mitochondrial protein synthesis. The isoaccepting Glu-tRNAs are detected by charging isolated yeast mitochondrial tRNAs with [ 3 H]glutamic acid using synthetase preparations obtained from mitochondria and fractionating the resulting aminoacyl- tRNA by RPC-5. Two distinct peaks of tRNA which accept glutamic acid are separated. Both fractions are isolated and shown to be gene products of mitochondrial DNA by nucleic acid hybridization. The hybridization of the two tRNAs to mitochondrial DNA indicates that the two tRNAs are coded by two different genes. Hybridization competition experiments and mapping experiments give further confirmation that these tRNAs are different in primary sequence. Subsequent experiments examine the codon responses of these two tRNAs in ribosome binding studies with synthetic oligonucleotides using the methods of Nirenberg and Leder. The tRNA eluting first from the RPC-5 column (GluI-tRNA) respond to oligonucleotides including the glutamic acid GAA and GAG codons. The tRNA eluting at higher salt (GluII-tRNA) did not bind to ribosomes in response to glutamic acid codons but did bind when an oligonucleotide including the glutamine codon CAA is used.

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.

We have isolated individual mitochondrial tRNAs from a petite mutant OI-P2-1 known to contain a limited subset of mitochondrial tRNA genes and have mapped these genes on the wild type genome of the yeast strains MH41-7B and D273-10B. To obtain DNA for fine structure mapping and DNA sequence analysis of these genes, we screened a yeast mitochondrial DNA-pBR322 recombinant bank with the isolated tRNAs. We report here the fine structure mapping of recombinant clones containing the tryptophan, formyl methionine and proline tRNA genes as well as the DNA sequence of the proline tRNA gene. The combination of restriction mapping and DNA sequence analysis has enabled us to locate these genes precisely on the wild type genome and to determine their direction of transcription.

Mitochondrial glutamyl-tRNA isolated from mitochondria of Saccharomyces cerevisiae was separated into two distinct species by re versed-phase chromatography. The migration of the two mitochondrial glutamyl-tRNAs (tRNA I Glu and tRNA II Glu ) differed from that of two glutamyl-tRNA species found in the cytoplasm of a mitochondrial DNA-less petite strain. Both mitochondrial tRNAs hybridized with mitochondrial DNA. Three lines of evidence demonstrate that mitochondrial tRNA I Glu and tRNA II Glu are transcribed from different mitochondrial cistrons. First the level of hybridization of a mixture of the two tRNAs to mitochondrial DNA was equal to the sum of the saturation hybridization levels of each glutamyl-tRNA alone. Second, the two mitochondrial glutamyl-tRNAs did not compete with each other in hybridization competition experiments. Finally the tRNAs showed individual hybridization patterns with different petite mitochondrial DNAs. Hybridization of the tRNAs to mitochondrial DNA of genetically defined petite strains localized each tRNA with respect to antibiotic resistance markers. The two glutamyl-tRNA cistrons were spatially separated on the genetic map.

DNA sequence analysis of mitochondrial genes that code for some mitochondrial proteins has suggested that the opal terminator, UGA, is used as a sense codon in mitochondria1–4. The complete sequences of both the yeast2,4 and human3 genes coding for cytochrome oxidase subunit II contain UGA codons in the reading frame. When the protein sequences predicted by these DNA sequences are compared with the known protein sequence of bovine mitochondrial cytochrome oxidase subunit II, there are regions of homology, in which UGA codons correspond to tryptophan residues. Therefore it has been suggested that UGA specifies tryptophan in the mitochondrial code. We have isolated a yeast mitochondrial tRNATrp and used it to locate the mitochondrial tRNATrp gene in pBR322-mitochondrial DNA recombinants. DNA sequence analysis of this gene revealed that the mitochondrial tRNATrp anticodon is 5′UCA3′. Because there is a U in the wobble position, this tRNA can recognize and insert tryptophan into a growing polypeptide chain in response to the nonsense codon UGA.

The TRM1 gene of Saccharomyces cerevisiae is necessary for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNAs. The DNA sequence of the TRM1 locus and the 5' ends of mRNAs expressed from this gene have been determined. The majority of the 5' ends map within a large open reading frame between two in-frame ATGs at positions +1 and +49. A small fraction of the 5' ends are located upstream of the first ATG. Both AUGs of the TRM1 mRNAs are used to initiate translation, and two forms of N2,N2-dimethylguanosine-specific tRNA methyltransferase, which differ by an amino-terminal extension of 16 amino acids, are made. Mitochondrial tRNAs are modified when the initiation of translation is limited to one or the other of the AUGs, suggesting that the amino-terminal extension is not necessary for import of the protein into mitochondria. Mitochondrial targeting information must, therefore, be located in a region of N2,N2-dimethylguanosine-specific tRNA methyltransferase that is found in both forms of the enzyme.

We have physically mapped the loci conferring resistance to antibiotics that inhibit mitochondrial protein synthesis (erythromycin, chloramphenicol and paromomycin) or respiration (oligomycin I and II), as well as the 21s and 14s rRNA and tRNA genes on the restriction map of the mitochondrial genome of the yeast Saccharomyces cerevisiae. The mitochondrial genes were localized by hybridization of labeled RNA probes to restriction fragments of grande (strain MH41-7B) mitochondrial DNA (mtDNA) generated by endonucleases EcoRI, HpaI, BamHI, HindIII, SalI, PstI and HhaI. We have derived the HhaI restriction fragment map of MH41-7B mit DNA, to be added to our previously reported maps for the six other endonucleases. The antibiotic resistance loci (antR) were mapped by hybridization of 3H-cRNA transcribed from single marker petite mtDNA's of low kinetic complexity to grande restriction fragments. We have chosen the single Sal I site as the origin of the circular physical map and have positioned the antibiotic loci as follows: C (99.5-1.Ou)--P (27-36.Ou)--OII (58.3-62u--OI (80-84u)--E (94.4-98.4u). The 21s rRNA is localized at 94.4-99.2u, and the 14s rRNA is positioned between 36.2-39.8u. The two rRNA species are separated by 36% of the genome. Total mitochondrial tRNA labeled with 125I hybridized primarily to two regions of the genome, at 99.5-11.5u and 34-44u. A third region of hybridization was occasionally detected at 70--76u, which probably corresponds to seryl and glutamyl tRNA genes, previously located to this region by petite deletion mapping.

We have cloned yeast mitochondrial DNA in the E. coli plasmid pBR322 by the poly(dA):poly(dT) tailing method and found that 62 of the 347 transformants obtained contain mitochondrial tRNA genes. In order to correlate the cloned mitochondrial tRNA genes with their gene products, we screened the recombinants with nick translated DNA isolated from petites known to carry choramphenicol (C), erythromycin (E), paramomycin (P), or oligomycin (0) markers as well as a limited subset of tRNA genes. A serine tRNA gene mapping in the OT region of the mitochondrial DNA was identified by hybridizing DNA from O 1 positive clones with [ 3 H]seryl tRNA. Other tRNA genes were identified by hybridizing [ 32 P]tRNA to Southern transfers of restriction enzyme digests of recombinant DNA molecules. Fragments selected for sequencing contained tRNA genes for two serine tRNAs, a phenylalanine tRNA, a glycine tRNA, a valine tRNA and an arginine tRNA. None of the tRNA genes appear to have intervening sequences, none have the CCA end encoded and all are surrounded by long stretches of very AT rich DNA.

Many nucleus-encoded mitochondrial enzymes differ in physical and chemical properties from analogous cytoplasmic enzymes, and it is therefore generally assumed that different genes encode analogous mitochondrial and cytoplasmic enzymes. However, our genetic studies show that for at least two different tRNA modifications, mutations in nuclear genes affect cytoplasmic as well as mitochondrial tRNAs. These studies utilize two yeast genes: TRM1 and TRM2 . trm1 cells do not have the enzyme activity necessary to methylate guanosine to N 2 ,N 2 -dimethylguanosine. trm2 is a new mutation that we describe here. trm2 cells are deficient in tRNA(uridine-5)methyltransferase, and hence contain tRNA lacking 5-methyluridine or ribothymidine. Other than lacking 5-methyluridine trm2 cells have no obvious physiological defect. These studies also show that the N 2 ,N 2 -dimethylguanosine and 5-methyluridine modifications are not added to tRNA in an obligatory order, and that 5-methyluridine is not required for removal of intervening sequences from precursor tRNA.

A mitochondrial specific RNase P is required to process 5' leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae. Experiments with a pair of mitochondrial pretRNAs(Asp) having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure. Asp-AU (75% AU leader) is cleaved by the mitochondrial RNase P while Asp-GC (39% AU) is not. Both are substrates for E. coli RNase P. Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5' leaders differ in secondary structure. It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.

Most components necessary for the biosynthesis of mitochondrial tRNAs are coded by nuclear genes, but one mitochondrial locus other than the tRNA genes themselves is required to make functional tRNAs in the yeast Saccharomyces cerevisiae. DNA sequence analysis of this yeast mitochondrial tRNA synthesis locus is reported here. This region of mitochondrial DNA is almost exclusively A+T-rich DNA with one G+C-rich element. Despite the unusual structure of the DNA in this region, we have demonstrated that it codes for a heretofore unidentified mitochondrial transcript about 450 bases in length. Since this RNA is the only RNA encoded by the tRNA synthesis locus, it must be the active agent of the locus. This RNA could either act autonomously through RNA-RNA interactions or as part of an RNA-protein complex to effect tRNA biosynthesis.

We have previously identified 14 aminoacyl tRNAs that are specified by yeast mitochondrial DNA (mtDNA). We now report four more amino acids (Arg, Cys, Trp, Thr) that acylate tRNAs which hybridize with mtDNA. Furthermore one of the two mitochondrial tRNAs that we had earlier demonstrated to be directly charged with glutamic acid responds to glutamine but not to glutamic acid codons. Thus Gln-tRNAGln appears to be formed by transamidation of a missense intermediate Glu-tRNAGln. This brings to 19 the number of amino acids which have corresponding tRNAs specified by mtDNA. Only tRNAAsn has not yet been shown to be a mtDNA transcript. We have also mapped the genes for the newly identified mitochondrial tRNAs, as well as several others that were previously identified but unmapped, by hybridization to the mtDNA of a series of petite deletion mutants. We now have ordered 20 mitochondrial tRNA genes (including two methionyl-tRNAs) wtih respect to the antibiotic resistance markers chloramphenicol (CR), erythromycin (ER), paromomycin (PR), and oligomycin I and II (ORI, ORII). Eighteen tRNA genes map between the C and E resistance markers. Only the serinyl-tRNA and glutamyl-tRNA genes are localized near the OI and OII resistance markers.

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30°. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.