Transcriptional Regulation of the Mitochondrial Genome of Yeast Saccharomyces cerevisiae"

The relative rates of transcription of several classes of the mitochondrial genes of the yeast Saccharomyces cerevisiae have been determined. The rates were meas- ured by pulse labeling whole yeast with [32P]04, isolating the total RNA, hybridization to single-stranded M13 DNA probes containing segments of the gene of interest, digestion with RNase A or T,, and separation of the protected fragment by gel electrophoresis. This analysis indicated that, among the genes analyzed, transcriptional promoters varied in strength by 20- fold while the rates of transcription varied by more than 50-fold. The strengths of the promoters of the genes were ordered: tRNAp > tRNAPhe > 14 S rRNA > 2 1 S rRNA > tRNAG'" > Oli- 1 > tRNACY". In addition, transcription rates were measured within polygenic transcription units. This analysis indicated that there was transcriptional attenuation within all the polygenic transcription units with the greatest attenuation factor being as much as 17-fold, occurring after the tRNAG'" and tRNAy genes. This analysis indicated that regulation of the rates of transcription in the yeast mitochondrial genome occurs by two distinct mecha- nisms, modulation of the rate of transcriptional initiation and attenuation of transcriptional elongation. The 75,000 bp' mitochondrial (mt) genome yeast Saccharomyces cereuisiae at four proteins of the electron transport chain (subunits 1, 2, and 3 of cytochrome oxidase and cytochrome b), three proteins of the ATP synthase (subunits 6,8, and 9), D273-10~ to prevent RNase degradation at this region of D-273-labeled RNA after hybridization with the M13 clone derived from ND157. Despite the considerable sequence divergence beyond this GC cluster, the next guanylyl residue does not occur for some 100 bases. This results in a fragment 492-bases long, protected from RNase T, digestion.

The relative rates of transcription of several classes of the mitochondrial genes of the yeast Saccharomyces cerevisiae have been determined. The rates were measured by pulse labeling whole yeast with [32P]04, isolating the total RNA, hybridization to single-stranded M13 DNA probes containing segments of the gene of interest, digestion with RNase A or T,, and separation of the protected fragment by gel electrophoresis. This analysis indicated that, among the genes analyzed, transcriptional promoters varied in strength by 20fold while the rates of transcription varied by more than 50-fold. The strengths of the promoters of the genes were ordered: t R N A p > tRNAPhe > 14 S rRNA > 2 1 S rRNA > tRNAG'" > Oli-1 > tRNACY". In addition, transcription rates were measured within polygenic transcription units. This analysis indicated that there was transcriptional attenuation within all the polygenic transcription units with the greatest attenuation factor being as much as 17-fold, occurring after the tRNAG'" and t R N A y genes. This analysis indicated that regulation of the rates of transcription in the yeast mitochondrial genome occurs by two distinct mechanisms, modulation of the rate of transcriptional initiation and attenuation of transcriptional elongation.
The 75,000 bp' mitochondrial (mt) genome of yeast Saccharomyces cereuisiae codes for at least four proteins of the electron transport chain (subunits 1, 2, and 3 of cytochrome oxidase and cytochrome b), three proteins of the ATP synthase (subunits 6,8, and 9), and a number of proteins involved in the expression of the genome. The genome also codes for a full complement of tRNAs, the large (21 S) and small (14 S) rRNAs, and a product involved in tRNA maturation (1,2). A single RNA polymerase encoded in the nucleus is responsible for the transcription of all of these genes.
The sites of transcriptional initiation in the mt genome have been mapped by in uitro capping analysis using the enzyme guanylyl transferase (3). This analysis indicated that there were a t least 19 unique sites of transcriptional initiation * This work was supported by Research Grant HL04442 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
dispersed throughout the genome. At each of the sites of initiation is the highly conserved sequence, ATATAAGTA, with the last A being the site of transcriptional initiation. Biochemical characterization of the mt promoter has been accomplished using the RNA polymerase purified from isolated mitochondria (4). Using bacterial plasmids containing this conserved nucleotide sequence, this polymerase is capable of selective transcription in uitro, with initiation occurring at the 3' end of the consensus nonanucleotide ( 5 ) . Further, deletion mutagenesis of these bacterial plasmids followed by i n uitro transcriptional analysis indicated that this conserved sequence was all that was required to promote specific transcription (6). Thus, by both biochemical analysis and consensus homology (7,8), this conserved sequence has been defined as the mt promoter for transcriptional initiation.
At most of the mitochondrial promoters there is an A at the +2 position, but there are a number of examples where transcriptional initiation occurs in uiuo at a divergent promoter sequence containing a T or G at this position. Divergences with a G or T at the -8 position rather than an A may also be transcriptionally active. Promoters serving as the sites of transcriptional initiation for the genes coding for the tRNAG'" (9), tRNACY" (lo), tRNAxpN (lo), and the 21 S rRNA (3,11) represent examples of these promoter variants. Furthermore, in petite strains, the steady state levels of these tRNA transcripts may differ considerably (3). These comparisons suggest that the strength of the promoter may determine the levels of different transcription products by regulating the frequency of transcriptional initiation.
There are a number of genes that do not abut on known promoter sequences. The genes are transcribed as parts of polygenic products, with transcription starting at the promoter of an upstream gene. Northern hybridization and/or SI experiments suggest that polygenic transcription occurs with the 21 S rRNA-tRNAx& (12,13), Oxi-3-AAP-01i-2 (14), Oli-1-tRNAS"-Var (15), tRNA?-TSL (16), and the tRNAG'"cytochrome b (9) genes, and within the tRNA cluster. With the exception of the gene for the tRNAxgN, the downstream genes of the other five gene clusters do not have the promoter sequence within the upstream intragenic space. Thus, transcriptional attenuation provides an additional possible mechanism of transcriptional regulation.
In this study, we report on the relative rates of transcription of a variety of genes included in polygenic and monogenic transcription units. The results indicate that the rates of transcription of promoter contiguous genes vary dramatically, with the greatest difference being about 20-fold. Further, there is transcriptional attenuation within all of the polygenic transcripts analyzed. Thus, transcriptional regulation of the mitochondrial genome occurs by at least two different mechanisms: 1) modulation of the rate of transcriptional initiation, and 2) transcriptional attenuation.

EXPERIMENTAL PROCEDURES
Derivation of the M I 3 Clones-The M13 probes used to analyze transcription are schematically illustrated in Fig. 1. Many of the clones are subclones of plasmids already described. The clone for the 21 S rRNA, mpYM21S, was obtained from digestion of the clone T5-208 (17) with the restriction enzymes Suu3A and HpaII. The DNA fragment (807 bp) was isolated and ligated into mp8 (18) digested with BumHI and AccI. The clone for the mitochondrial 14 S rRNA, mpYM132b, was obtained by digestion of the plasmid, pYM132b, with the restriction enzymes Hind111 and EcoRI. The DNA fragment (720 bp) was isolated and ligated into mp9 (18) digested with the EcoRI and HindIII. The plasmid, pYM132b, was obtained by digesting pYM132a (17) with Hue111 and HindIII. The DNA fragment (720 bp) was isolated and ligated with the vector, pUR250, digested with Hue111 and HindIII. The plasmid was made blunt with T, DNA polymerase and ligated again. The resulting clone was found later to have an additional 190-bp fragment inserted downstream of the correct insert. The probe for the Oli-1 transcripts, mpYMK9, is a subclone of the plasmid pK9 (19), obtained by digesting pK9 with the restriction enzymes EcoRI and PstI. The 892-bp insert was isolated and ligated with mp9 digested with the same restriction enzymes. The 5 S rRNA probe, mpY5SA, was obtained by isolating the B restriction fragment of the EcoRI digestion of the rRNA cistron. This B fragment was obtained by digesting the clone, pYrB (20), with EcoRI. The B restriction fragment was then digested with the restriction enzyme TugI. The resulting 416-bp fragment was isolated and ligated with mp8 digested with AccI. After transformation and screening, only deletion mutants of the expected product were obtained.
The clone, mpY5SA, contains only 22 bases of the 3' end of the 5 S rDNA coding region with the DNA 5' to this region deleted. The clone, mpY18S5.8, was obtained from digestion of the plasmid pYaR12' (20) with the restriction enzyme EcoRI. The DNA fragment (643 bp) was ligated into mp8 (18) digested with EcoRI. The probe for the cytochrome b RNA, mpYME6B2, was obtained from digestion of MH-41 mitochondrial DNA with the restriction enzyme EcoRI. Restriction fragment 6 (21) was isolated, digested with the enzyme BglII, the 1402-bp fragment isolated, and ligated with mp8 digested with BamHI and EcoRI. The probe for Oxi-3, mpYMElO, was also obtained from MH-41 mitochondrial DNA. Restriction fragment 10, after digestion with EcoRI, was isolated and ligated with mp8 digested with EcoRI. The probe for the transcripts of the gene coding for subunit 6 of the ATPase (Oli-2), mpYMOII, was derived from MH-41 mitochondrial DNA. The EcoRI restriction fragment 7 of MH-41 mtDNA was isolated and digested with HinfI. This was ligated with mp8 digested with EcoRI and HincII, the plasmid was made blunt with Klenow fragment, then ligated. After transformation, the clones were screened for the 355-bp insert. The probe for the tRNAG'", mpYME, was obtained from MH-41 mtDNA. The restriction fragment containing the gene for the tRNA was isolated after digestion with the enzyme Sau3A. The fragment was digested with the enzyme TuqI, and the 235-bp digestion product was isolated and ligated with mp9 digested with AccI and EcoRI. The three remaining probes have all been obtained from D273-108 mtDNA. Two of these are subclones of clones described elsewhere. The clone mpYMF is a subclone of pYmpFl (22). The mitochondrial insert was obtained by digesting pYmpFl with the restriction enzyme EcoRI and HindIII. The insert (680 bp) was ligated into mp8 digested with the same enzymes. The clone, mpYMCT2, was obtained by digesting the plasmid pYmpCl (22) with EcoRI and HindIII. The mitochondrial insert (571 bp) was isolated and ligated into mp8 digested with the same enzymes. The M13 plasmids were all transformed into JMlOl (23 Isolation of the Single-strunded Phage DNA-Single-stranded DNA was obtained by inoculating 1 ml of a preculture of JMlOl into 100 ml of YT media with 0.01 ml of phage supernatant. After growth for 6 h at 37 "C, the cells were centrifuged for 10 min at 5000 X g. The supernatant was centrifuged a second time as above. The supernatant was carefully removed and the phage precipitated by adjusting the solution to 3.3% polyethylene glycol (SOOO), 0.42 M NaC1. The phage were isolated by centrifugation at 5000 X g for 20 min and the sediment dissolved in TE buffer. The suspension was extracted three times with phenol/chloroform/isoamyl alcohol, and the DNA was precipitated after adjusting the solution to 0.3 M NaOAc and 66% ethanol and incubating it at -80 "C for 30 min. The DNA was isolated by centrifugation at 9000 rpm in the SS-34 rotor and the sediment washed once with cold 66% ethanol. The DNA was dissolved in TE buffer to a concentration of 1 mg/ml. 3Dr. Nancy Martin, Dept. of Biochemistry, The University of Texas, Dallas, TX.
RNA was removed from the preparation by adjusting the solution to 0.3 M NaOH and incubating it at 37 "C for 30 min. An equivalent of glacial acetic acid was added, and the DNA was recovered by ethanol precipitation at -80 "C for 30 min. The DNA was isolated by centrifugation, washed with 66% ethanol, and dissolved in TE buffer as described above. The DNA solution was extracted with chloroform as necessary until the APm nm/A280nm ratio was 2.0. The concentration of single-stranded DNA was calculated using 1 ODzm equal to 40 pg/ ml.
Labeling RNA by in Vitro Trans~ription-[~*P]UMP was incorporated into RNA by the mitochondrial in vitro transcription system (17). The polymerase was incubated at 28 "C for 10 min with the vector pYM132b digested with either the restriction enzyme ClaI or PuuII. The DNA was digested with DNase, and the unincorporated nucleotides were removed using the spin column (26) with the resin Bio-Gel P6 equilibrated with TE buffer containing 0.3 M NaOAc and 0.2% sodium dodecyl sulfate. To the RNA, 5 pg of tRNA was added, and the RNA was precipitated with three volumes of ethanol and incubation at -80 "C for 30 min. The RNA was recovered by centrifugation, as above, and dissolved in T E buffer.
Lubeling and Isolation of Yeast RNA-The yeast strain, D273-10B (ATC no. 25647), was grown at 28 "C to mid-log phase (2 X lo7 cells/ ml) in phosphate-depleted media (27) consisting of 2% peptone, 2% galactose, and 1% yeast extract. The cells (4 ml), were washed three times with media lacking phosphate. Media lacking phosphate consisted of 15 mM PIPES, pH 5.9, 2% galactose, all the salts, minerals, amino acids, adenine, and uracil at the concentrations described but omitting any phosphate salts. The washed cells were suspended in 2 ml of 2 X media lacking phosphate, 1 ml was removed and added to 1 ml of carrier free [32P]04 in dilute acid (8 mCi/ml) and rotated at 28 "C for 10 min. 1 ml was removed, and the remaining 1 ml was rotated for an additional 10 min. The cells were placed on ice, centrifuged for 5 min in an Eppendorf centrifuge, and washed with cold water. The cells were digested at 37 "C for 1.5 min with 10 mg/ ml zymolyase in 1.2 M sorbitol, 50 mM potassium phosphate, pH 7.8, 1 mM EDTA, and 2-mercaptoethanol (1/500, v/v). The spheroplasts were centrifuged for 10 min in the Eppendorf centrifuge and the supernatant removed. To the sedimented spheroplasts, 0.1 ml of glass beads (250-pm diameter) and 0.4 ml of 10 mM Tris-C1, pH 7.4, 5 mM EDTA, 0.1 M NaCl, and 0.2% sodium dodecyl sulfate were added and the suspension agitated vigorously with a vortex shaker until the suspension appeared homogeneous. This was extracted three times with 0.4 ml of phenol/chloroform/isoamyl alcohol (50:49:1). The aqueous layer was adjusted to 2 M ammonium acetate and the nucleic acids precipitated with three volumes of ethanol and incubated at -80 "C for 30 min. The nucleic acids were isolated by centrifugation for 15 min in the Eppendorf centrifuge and washed once with cold 66% ethanol. The nucleic acids were dried in uucuo and then dissolved in TE buffer (0.1 ml) containing 10 mM MgC12. The DNA was removed by digesting the nucleic acids with DNase (0.03 mg/ml, RNase free (28)) at 37 "C for 10 min. The solution was adjusted to 0.2 ml with TE, extracted with phenol/chloroform/isoamyl alcohol (0.2 ml), precipitated with ethanol, washed with 66% ethanol, and dissolved in TE buffer (0.1 ml) as described above.
Hybridization and Digestion of the RNAIDNA Hybrid-The hybridization was performed in 0.05 ml of a buffer that consisted of 0.6 M NaC1,50 mM bicine, pH 7.8, and 1 mM EDTA. The RNA and DNA were added together, heated to 100 "C for 3 min, then incubated for 12 h at 60 "C, unless noted otherwise. Typically, 2 pg of M13 DNA was used per reaction (see below). Upon completion of the hybridization, 50 mM bicine, pH 7.8, 1 mM EDTA, and 0.1 pg/ml of wheat germ tRNA was added (0.05 ml). Unless noted otherwise, RNase TI (300 units, 300 units/pl) was added and the solution incubated for 30 min at 37 "C. For some reactions, in addition to RNase TI, RNase A (0.015 units, 0.0075 unitslpl) was added and incubated as above. After digestion with RNase, proteinase K was added (40 pglml) and the reaction mixture incubated at 37 "C for 60 min, then moved to room temperature.
The RNA samples were either precipitated with ethanol and 5 pg/ ml carrier tRNA or purified on a nucleic acid chromatography system column. In the latter case, the solution was adjusted to 0.5 M NaCl with 2 M NaCl in TE buffer and loaded onto a nucleic acid chromatography system mini-pre-pack column (Bethesda Research Laboratories) equilibrated with 0.5 M NaCl in TE buffer. The column was washed with 0.5 M NaCl in TE (0.3 ml), followed by 1.0 M NaCl in TE (0.3 ml), then eluted with 1.5 M NaCl in TE (0.4 ml). To this, carrier tRNA (5 pg) and 1.0 ml of ethanol was added. The solution was placed at -80 "C for 40 min and the RNA/DNA isolated by centrifugation in an Eppendorf centrifuge for 15 min. The sediment was dissolved in 2 M NH,OAc in TE buffer and reprecipitated with ethanol and 2 M NH4OAc. The sediment was washed once with 66% ethanol.
For samples whose RNase-protected fragments were less than 100 nucleotides long, two additional steps were included to remove small oligonucleotides. After digestion with proteinase K, the samples were adjusted to 0.5 M NaCl in TE (0.5 ml), and the samples were incubated for 1 h at 60 "C before loading onto the NACS column, as above. The column was washed as above, but eluted with only 0.15 ml of 1.5 M NaCl in TE. The eluant was immediately placed on a 1 ml spin column (26) containing Sephacryl S-300 equilibrated with 0.5 M NaCl in TE buffer. The samples were ethanol precipitated and washed as described above. These additional steps were sufficient to eliminate most of the small oligonucleotides that were not specifically hybridized to the M13 probe and otherwise obscured the signal.
To ensure that all cognate RNA was hybridized with the added probe, experiments were performed with increasing amounts of probe. The results of these experiments are recorded in detail in an accompanying manuscript.' 1 pg of the M13 probe for the mt-rRNA represented about 30-fold molar excess over the mt-rRNA isolated from lo6 cells. This number was derived from the number of copies of the mt-rRNA determined from cells grown under derepressive conditions? For all experiments, increasing the concentration of the probe resulted in no further increment in the relative signal of the protected RNA fragment. Typically 2 pg of the M13 DNA was used per reaction, per 5 X lo5 cells representing a molar excess of more than 100-fold.
In the case of the probe for tRNA&, the size of the RNA/DNA hybrid is only 38 bases. Despite the small size of the hybrid, the probe concentration appeared to be sufficiently high to drive the hybridization to apparent completion. However, in view of the possibility that hybridization might be incomplete for small RNA/DNA hybrids, an additional experiment was undertaken. A probe for the cytoplasmic 5 S rRNA which only provides a 22-nucleotide RNA/DNA hybrid was used to determine the concentration of the 5 S rRNA relative to the 18 S (228-base pair hybrid) and 5.8 S (89-base pair hybrid) rRNA.
For this analysis, these RNAs were labeled by growth in precursor 32P, i.e. steady state labeling. Assuming that these species are present at equal molar levels, our analysis showed that using the 5 S probe, this RNA could be underestimated by no more than 35%. This sets the outside limit for the underestimation of transcription of the Gel Electrophoresis of the Samples-The samples were dried in uacuo, then dissolved in 95% formamide, 2 mM EDTA, and 0.02% bromphenol blue, xylene cyanol. The samples were denatured at 100 "C for 3 min immediately prior to electrophoresis through either a 5 or 10% polyacrylamide sequencing gel containing 50% urea (29). The bands were visualized with Kodak XAR5 film with or without a Dupont Lightning Plus intensifing screen at -80 "C.
Quantitation of the Autoradiogram-The intensity of the autoradiogram was determined either by densitometry or by elution of the silver grains followed by measurement of the light scattering (30). In the former case, an LKB laser scanning densitometer interfaced with a Hewlett-Packard 3390A integrator was used. For both procedures, various exposures were used to ensure that all data was obtained from signals that were not overexposed.
Materials-The restriction enzymes were purchased from either Bethesda Research Laboratories, Boehringer Mannheim, or P.L. Biochemicals. The nucleotides, proteinase K, and RNase A were products of P.L. Biochemicals while RNase TI was obtained from Worthington. The NACS pre-pack mini-columns were purchased from Bethesda Research Laboratories. These were regenerated by washing them with 4 M NaC1, 1 M NaOH followed by 1 M NaCl in TE buffer. After regeneration the columns were dried in an oven and stored at room temperature. The yeast extract and peptone were obtained from Gibco, and the galactose and glucose used for the media were obtained from Sigma. tRNAxPN.

RESULTS
The method employed for the analysis of the rates of transcription depends on the selective protection of RNA from RNase digestion by hybridization to single-stranded DNA probes. This procedure uses M13 clones, containing the region of interest, in the orientation such that the single- The DNA is used to hybridize to pulse-labeled RNA, followed by digestion with the single-stranded selective RNases, A or T,, and the RNA/DNA hybrid is isolated by column chromatography, denatured, and separated by gel electrophoresis. The amount of the selected radioactive RNA fragment protected from the RNase digestion, is a measure of the level of that specific RNA. This procedure is specific, quantitative, and applicable to all the RNA types, rRNA, mRNA, hnRNA and tRNA. The M13 Probes Used in This Analysis-The M13 clones used in this study and the predicted sizes of the major hybridized fragments protected from RNase (RNase A and/or Tl as indicated in the figure legend) are illustrated in Fig. 1. The clone, mpY5SA, contains a 22-nucleotide portion of the cytoplasmic 5 S rDNA. Upon hybridization of the phage DNA of this clone to total cytoplasmic RNA, a 23-nucleotide protected fragment is expected. The different lengths of the 5 S rDNA insert (22 nucleotides) and the RNA fragment protected (23 nucleotides) relates to the base sequence of the RNA and the specificity of the RNAse used to digest the unhybridized RNA. In the case of the 5 S RNA, there is an additional G overhang. The mitochondrial clones contain regions coding for the 21 and 14 S rRNA (mpYM21S and mpYM132b), the 9 S RNA (NS6), subunits 6 (Oli-2) and 9 (Oli-1) of the ATP synthase (mpYMOII and mpYMKS), cytochrome b (mpYME6B2), subunit 1 of cytochrome oxidase (mpYMElO), and tRNA glutamic acid (mpYME), phenylalanine (mpYMF), N-formyl methionine (mMet4), and cysteine and threonine-2 (ACN) (mpYMCT2). The 9 S RNA is a product of the tRNA synthesis locus (TSL) which is involved in processing the 5' ends of the precursor tRNA molecules and postulated to have RNase P-like activity (16,25). The clone, NS6, was derived from a petite strain of MH-41. The nucleotide sequence in this region of this petite clone diverges from the corresponding sequence of the grande strain used in this study, D273-10~. Because of this mismatch, hybridization of D-273 RNA to the NS6 clone yielded two fragments (296 and 140 nucleotides in length) that are protected from RNase digestion, as illustrated. In addition, when RNase T1 is used to digest the primary transcript hybridized to the NS6 clone, fragments of 205 and 492 nucleotides in length are p r~t e c t e d .~ The 14 S rRNA probe also protects two digestion products. The 14 S rRNA is synthesized as a 14.5 S precursor with approximately 80 additional nucleotides at the 5' end. The rate of processing the 5' end sequence will determine the relative product amount derived from the precursor (381 nucleotides in length) and from the mature (301 nucleotides in length) 14 S rRNA transcript.
The locations from which each of the M13 clones are derived, and the sites and approximate extents of expected RNA hybridization are illustrated in Fig. 2. Included are the pertinent sites of transcriptional initiation. The clones, serving as probes of the 21 S rDNA, 14 S rDNA, Oli-1, tRNACY", and the t R N A p genes, overlap the transcriptional promoter in each case. This allowed an assessment of the fidelity of transcriptional initiation as well as of the rates of transcription. In four instances, probes are within the upstream and The petite strain, ND157, from which this clone was derived diverges in its nucleotide sequence around 80 nucleotides downstream of the mature 3' end of the TSL. However, this divergence is within a GC cluster, which shares enough homology with D273-10~ to prevent RNase degradation at this region of D-273-labeled RNA after hybridization with the M13 clone derived from ND157. Despite the considerable sequence divergence beyond this GC cluster, the next guanylyl residue does not occur for some 100 bases. This results in a fragment 492-bases long, protected from RNase T, digestion.
downstream regions of polygenic transcription units. This occurs in the polygenic transcription units of the 21 S rRNA and the tRNAx&, the t R N A p and the TSL, the Oxi-3 and the Oli-2, and the tRNAG'" and cytochrome b genes. Thus, with these probes we can determine whether attenuation occurs during transcription in these polygenic units.
Demonstration of Protection of RNA in an RNAIDNA Hybrid from RNase Digestion-Both RNase A and T1 selectively degrade single-stranded RNA. However, RNase A will degrade RNA in an RNA/DNA if used at a high enough concentration. This is illustrated in Fig. 3. In this experiment, RNA was labeled in vitro by transcription of a bacterial clone containing a portion of the 14 S mitochondrial rRNA gene cut with the restriction enzyme PuuII, whose sites are in the DNA of the bacterial plasmid. The labeled RNA was hybridized with mpYM132b, which shares the mitochondrial sequences within the transcription product, digested with different concentrations of RNase, and the products separated by gel electrophoresis. The transcription reaction yields a fragment of 720 nucleotides while the size of the protected fragment is predicted to be 384 nucleotides long. Lanes 1-6 illustrate digestion of the RNA/DNA hybrid with RNase A (0.0015-150 units). At the lowest concentration of RNase A, there was incomplete digestion of the 720-nucleotide run-off transcript, but still a considerable amount of the correct 384-nucleotide product was present. At 0.015 units of enzyme, there was almost complete conversion of the run-off product to the size predicted for the RNA/DNA hybrid, 384 nucleotides. At higher concentrations than this, however, there was degradation of the RNA in the RNA/DNA hybrid. The degradation of the RNA in the DNA hybrid did not occur with the enzyme RNase T1. Lanes 7-12 illustrate the digestion of the RNA using RNase T, ranging from 0.003-300 units. Using this enzyme, the theoretical size of the protected fragment is 382 nucleotides in length. Even at 300 units of enzyme, there was little degradation of the RNA in the hybrid with seemingly complete conversion of the 720-nucleotide transcript to the fragment 382 nucleotides long. Thus, only in a very narrow range of enzyme concentration, was RNase A selective for the single-stranded RNA and still retained enough activity to completely digest the single-stranded RNA. This limited its usefulness and hence, RNase T1 was used for most of the experiments.
Denaturation of the RNA/DNA hybrid prior to digestion with RNase Tl abolishes the 382 nucleotide long digestion product. This experiment is shown in lane 13. Furthermore, no products are seen if an M13 probe is used which contains no homology with the RNA substrate (data not shown). Therefore, the products obtained after hybridization and digestion with RNase are those oligonucleotides engaged in an RNA/DNA hybrid.
The amount of time necessary for complete hybridization was also determined. This was done in a similar fashion except that the run-off transcription product was from the site of initiation to the ClaI site, 373 nucleotides in length. This ClaI site is included within the insert in mpYM132b, and thus the whole transcript should be protected. Any degradation of the transcript by RNase after hybridization would be indicative of incomplete hybridization. The run-off transcript was hybridized for 1 h with mpYM132b, half of the reaction mixture was precipitated with ethanol, while the remaining half was digested with RNase T,. The products of these reactions are shown in lanes 14 and 15, respectively. In only 1 h, 80-90% of the total transcript was hybridized to the DNA as indicated by its resistance to RNase. Longer incubations allowed for quantitative protection of the RNA from RNase T1. Analysis of in Vivo-labeled RNA: Determination of the Relative Rates of Transcription-The use of in vitro-labeled RNA was useful for the identification of the correct conditions for hybridization and RNase digestion, but with in vivo-labeled RNA the situation was more complex. Upon labeling the whole yeast with [32P]04, a large amount of material was labeled that was resistant to RNase, precipitated with ethanol, and obscured the signal. To circumvent this problem, the RNA/DNA hybrid was purified on a nucleic acid chromatography system column after digestion with RNase. This rapid purification was necessary and sufficient for the isolation of the RNA/DNA hybrid.
The rates of transcription were assessed by pulsing yeast with ["P]04. Fig. 4 illustrates an autoradiogram of the fragments protected from RNase TI after hybridization to M13 probes and separation by gel electrophoresis. The RNA was intensity of the major protected bands of the 21 and 14 S rRNA, the rates of their transcription are comparable. This is consistent for the 10-and 20-min labeling times. Secondly, the rate of processing of the 5' end of the 14.5 S RNA is extremely slow, with only a little of the 301-nucleotide product, the product of the mature 5' end, present in yeast labeled for 10 min. Even after labeling for 20 min, the majority of the product is represented by the precursor product, 381 nucleotides in length. It is notable that there was no evidence of a protected band corresponding to the full length of either the 21 or 14 S rRNA probes. Such a product would occur only if there was transcription initiated upstream of the known promoter. Hence the initiation of transcription of these two genes must be at the previously identified promoters (3). The second lane illustrates the products protected from RNase by hybridization with the 14 S rRNA and the Oli-1 probes. The transcription from the 14 S rRNA gene is clearly greater than that from the Oli-1 gene, in the yeast labeled for both 10 and 20 min. The major digestion product for the Oli-1 RNA is 244 nucleotides long, but a minor fragment of about 255 nucleotides in length is also present. This product is present in the same proportions with either pulse-labeled RNA or steady state-labeled RNA (data not shown). The origin of this larger band is not known since there is neither a known transcriptional initiation site nor a consensus promoter sequence to account for it. There is a second consensus promoter sequence about 80 nucleotides downstream from the major promoter, but this second promoter does not appear to be active under these conditions. The predicted digestion product (164 nucleotides long) of the RNA transcript originating from this second promoter is not seen.
Hybridization of the Oli-1 and Oli-2 probes with labeled RNA protects the products shown in lane 3. Transcription of the Oli-1 gene was not only more frequent than that of the Oli-2 gene but also more frequent than that of other protein genes. Lane 4 shows the products protected from RNase digestion after hybridization with the Oli-2 and the Oxi-3 probes. These genes are transcribed as part of a single unit (Fig. 2). This experiment reveals a significant difference in the abundance of the fragments protected from RNase digestion by hybridization with the Oxi-3 and with the Oli-2 probes, with the Oxi-3 being more abundant.
Lane 5 shows the fragments protected after hybridization with the Oli-1 and the cytochrome b probes. The fragment protected by the cytochrome b probe (416 nucleotides in length) was not evident in this expocure with the RNA from yeast labeled for 10 min, but was visible in the RNA of yeast labeled for 20 min. Despite the fact that the cytochrome b probe protects an RNA fragment almost twice as long as the Oli-1 protected fragment, a much lower intensity of the signal was observed. Hybridization of labeled RNA to the Oli-1 probe and the probe for the tRNA synthesis locus protects the fragments from digestion with RNase as illustrated in lane 6. The fragments that should be protected from RNase after hybridization of the mature RNA from the tRNA synthesis locus are 296 and 140 nucleotides long, neither of which was observed. Instead, only fragments 492 and 205 nucleotides long are present. These correspond to the sizes predicted for the digestion products of the primary transcript protected by hybridization to the NS6 probe. The absence of either the 296-or the 140-nucleotide long protected fragments indicates that both the 5' and 3' processing of the primary transcript of the tRNA synthesis locus were extremely slow.
The analysis of the transcription of the mitochondrial genome was extended to the tRNA genes. For this study, slight modifications were made in the procedure to separate the small protected fragments from the nonspecific degradation products of the non-hybridized RNA (see "Experimental Procedures"). For these experiments two internal control probes were added to each hybridization reaction mixture, the Oli-1 probe and the probe for the cytoplasmic 5 S rRNA. The Oli-1 probe allowed this analysis to be compared to that of the other mitochondrial genes and the 5 S probe allowed a qualitative comparison of the rates of transcription of the mitochondrial genes with that of the cytoplasmic rRNA. As before, the RNA was labeled for 10 and 20 min. Fig. 5 illustrates the results of the experiment. In lane 1 are shown the transcription products protected by hybridization with the probes for the tRNAG1" and tRNAPh' genes. In addition to the predicted products for the glutamic acid and phenylalanine tRNAs (58 and 73 nucleotides long, respectively) the products of the Oli-1 RNA and the 5 S rRNA (244 and 23 nucleotides) were also present. For both the 10-and 20-min pulses, the transcription of the tRNAPh' gene was greater than that of the tRNAG1" gene. No other major products were observed, not even those that would correspond to the precursor tRNAs. Thus, the processing of these tRNAs was extremely rapid, under these conditions.
The second lane shows the products protected from diges- tion by hybridization with the probes for the tRNAPh' and the tRNA,"'" genes. The major predicted fragments of the tRNAPhe and the t R N A p (57 nucleotides in length) RNAs were present. In addition there was a significant ladder below the 57nucleotide fragment. This ladder was only seen when the t R N A p probe was used and then only occurred with RNA that was pulse labeled. Using steady state-labeled RNA, the t R N A p probe protected only a single fragment with a length of 57 nucleotides (data not shown). Though the cause of this ladder is not clear and its presence did seem to interfere slightly with the analysis, the background could be subtracted from the signal obtained by densitometric scanning. The gene for the tRNAyt is part of a polygenic transcription unit which includes the tRNA synthesis locus. As noted above, the processing of the 5' and 3' ends of the primary transcript from the tRNA synthesis locus is extremely slow. The secondary structure of this precursor RNA molecule may have been responsible for this ladder as suggested by its absence with steady state-labeled RNA.
The products protected by hybridization with the probes for the tRNAx& and tRNACy" genes are illustrated in lane 4. The predicted protected fragments of the tRNAx& (38 nucleotides) and the tRNACYs (59 nucleotides) were readily seen Transcriptional Regulation of the Yeast Mitochondrial Genome only in the sample from the yeast labeled for 20 min. A longer exposure showed the protected bands in samples from the yeast pulse-labeled for 10 min. The relative intensity of the two protected fragments suggests that the tRNAxpN gene was transcribed a t a much greater rate than the tRNACy" gene, even though only 500 nucleotides separate these two genes. The final lane shows the control fragments protected by hybridization with only the probe for the cytoplasmic 5 S rRNA. The autoradiograms were scanned by laser densitometry and the area integrated electronically. To ensure linearity of photographic plate response, as well as maximum sensitivity, several exposures of each autoradiogram were analyzed. The results of this are shown in Table I. All transcription rates reported in this table are related to the normalized rate of transcription of the mt 14 S rRNA gene. The relative rates of transcription of the genes were comparable when determined for yeast labeled for 10 and 20 min. However, the two determinations of the transcription rates for the cytochrome b and the tRNAG'" genes differed. In these two cases, the relative transcription rates appeared to increase from the 10-to the 20-min pulse time. This discrepancy occurred only for these gene products, which are transcribed as a polygenic product (9). The relative rate determined for the 20-min pulse is considered to better represent the in vivo rate of transcription, because of its higher value.
The relative rates of transcription of the remaining genes were concordant whether determined with the yeast labeled for 10 or for 20 min. The apparent rates of transcription varied 50-fold, ranging from the relative rate of 1.7 for the tRNA? gene to 0.03 for the cytochrome b gene (20-min pulse). The rates of transcription of the genes for the large and small rRNA were similar though the rate of transcription of the 14 S rRNA gene was consistently 30-40% greater than that of the 21 S rRNA gene. Of the four protein genes analyzed, the Oli-1 gene was transcribed at the highest rate, being about 8-fold higher than that of the Oli-2 gene and 10fold higher than the rate of transcription of the cytochrome b gene. All tRNA genes were rapidly transcribed, with the exception of the tRNACy" gene.  For the genes whose transcriptional promoters are located just upstream of the genes, the differences in the relative rates of transcription are probably attributable to differences in promoter strength. Thus, the tRNA,"t promoter was strongest and the tRNACy" promoter the weakest differing from one another by about 20-fold. The promoter strengths of the remaining genes, the 21 S rRNA, 14 S rRNA, Oli-1, tRNAPhe, and the tRNAG'" genes, were all similar. The rate of transcription of the Oxi-3 gene was about l/10 that of the 14 S rRNA, despite the fact that their promoter sequences were nearly identical. Since the probe used for this study was at the end of the long intron containing gene, this rate may not accurately reflect the effective strength of the promoter (see below).
The remaining genes, the TSL, cytochrome b, Oli-2, and the tRNAxFN, are all thought to be downstream members of polygenic transcription units. With the exception of the tRNAxFN gene, there is not a neighboring homologous promoter sequence in front of these genes. In the case of the gene, there is a divergent potential promoter sequence with changes at the -8 (G) and +2 (T). There is little evidence that this promoter is active in uiuo, and it is extremely weak when assayed in vitro (22). Thus, the major contribution to the transcription of the tRNAxFN gene probably originates as a cotranscription product from the 21 S rRNA promoter. The differences in the apparent rates of transcription of the downstream genes and the upstream genes of polygenic transcription units reflect the amount of transcriptional attenuation.
Transcriptional Attenuation-The experimental basis for the transcriptional attenuation determined in this study is summarized in Fig. 6. There was just a small difference between the rates of transcription of the 21 S rRNA and the tRNAxFN gene. However, transcription of the tRNACY" gene is at an 8-fold lower frequency than of the This low level of transcription is compatible with transcriptional initiation from the promoter directly in front of the tRNACYs gene and would involve transcriptional termination after the tRNAxFN gene. Alternatively, a small amount of transcription of the tRNACy" gene might derive in part from a polygenic transcript originating from the 21 S rRNA gene promoter.
Thus, the 8-fold lower frequency of tRNACY" transcription would represent the minimal attenuation factor. Large attenuation factors were also observed for the transcription of the TSL and the cytochrome b genes. These downstream genes were transcribed 17-fold less frequently than their upstream partner genes. The attenuation factor for the Oli-2 gene was less pronounced. This gene was transcribed at 2-3-fold lower frequency than the Oxi-3 gene. Among these five examples, the strongest attenuation occurs with the polygenic transcription units having a tRNA gene as the upstream element.

DISCUSSION
We have measured the relative rates of transcription of mitochondrial genes in yeast S. cereuisiae. To undertake this study, we have developed a rapid and sensitive method which employs M13 clones containing a portion of the gene of interest. The clones are constructed so that the M13 singlestranded phage DNA is complementary to the RNA transcription products. After pulse labeling the cells with [32P]04, the total nucleic acids are isolated, hybridized with one or more M13 clones, digested with the single-stranded selective RNases A or TI, the RNA/DNA hybrid purified, and the RNA fragments separated by gel electrophoresis and visualized by autoradiography. This method has several advantages over dot blot hybridizations. First, the hybridization is done in  Fig. 2) within polygenic transcription units analyzed in this study. The arrows indicate the major known transcription units with the relative rates of transcription at the point measured shown below. The attenuation factor is the fraction of the higher transcription rate/lower transcription rate.
solution allowing for rapid and near quantitative hybridization. Second, more than one probe can be added to the same hybridization reaction. This permits the rate of transcription of a number of genes to be assessed simultaneously and also allows the addition of an internal control from which the rates of transcription of several genes can be gauged. Finally, the gel electrophoresis of the RNase-protected bands permits their separation from nonspecifically bound labeled RNA, thereby providing a low background.
The relative rates of transcription determined for genes which have closely neighboring transcriptional promoters, the 21 S rRNA, 14 S rDNA, tRNAcy', tRNAPhe, and the tRNA? genes, can be considered to reflect the relative strengths of the respective promoters. These results indicate that the order of the relative promoter strengths are: t R N A p > tRNAPh" > 14 S rDNA > 21 S rDNA > tRNAG1" > Oli-1 >> tRNAcya. This rank order is in close agreement with that determined using the in vitro transcription system and bacterial plasmids containing some of these promoters (22). The analyses of the rates of transcription of the tRNA genes could be complicated by the turnover of the 3' terminal CCA trinucleotide. Because these nucleotides are added to the tRNA post-transcriptionally, it is possible that their turnover and consequent incorporation of 32P during the labeling might lead to a spuriously high estimate of the transcription rates. With cytoplasmic tRNA the terminal CCA and particularly the adenine has been shown to turn over significantly (31,32). For a number of reasons, we do not believe that the turnover of the terminal CCA trinucleotide significantly contributed to our estimation of the rates of transcription. First, the rates of transcription as determined in vivo correlated very well with the rates of transcription determined in uitro.
This correlation was demonstrable despite the fact that some of our probes only protected the 5' end of the tRNA (tRNAG1" and tRNACy") while the others protected the 3' end of the tRNAs. Second, there is also a strong correlation between the rates of tRNA transcription determined here and the levels of the tRNAs determined after steady state labeling of the RNA4 when CCA trinucleotide turnover should not complicate the interpretation. Third, similar results were obtained when the RNA/DNA hybrids were digested with RNase T, alone as when RNases T, and A were used together. Under the former conditions, the terminal A will not be removed from any of the tRNA hybrids for tRNA Cys, F-Met, or Phe. In the case of the tRNACy*, the terminal CCA is left undigested using only RNase T, while it is completely removed from the hybrid using the combination of RNases A and T,. The similar results obtained from the apparent transcription rates of tRNAs regardless of whether the CCA is retained or not indicates that the independent turnover of the 3' CCA does not significantly influence our estimates of tRNA transcription rates in the mitochondria. The differences in the relative strength of the promoters studied here can be correlated with the sequence in and around the consensus promoter. Fig. 7 shows the nucleotide sequences of the promoters assessed in this study as well as some promoter-like sequences which are apparently nonfunctional i n vivo and are either extremely weak or nonfunctional i n vitro. The major differences correlated with the nucleotides found at the -8 and the +2 position. Those promoters that are extremely weak, e.g. the tRNAcys, or inactive, as in the promoter-like sequences downstream of the active promoters of the Oli-1 and the tRNA? genes, have a T at the +2 position. With a T residue at the -8 position, the promoter is stronger than when an A residue occupies this promoter position as in the promoters for the tRNA,"" and the tRNAPh" genes. The promoter for the tRNACys has a T residue at both the -8 and +2 position with barely measurable promoter activity. This is in contrast to both the inactive promoters that have T residues at the +2 position and A residues at the -8 position. The importance of the +2 position has been recently emphasized in a study involving site specific mutagenesis of the promoter sequence (33).
The apparent relative rates of transcription of the upstream and downstream genes of the polygenic transcription units indicated that transcriptional attenuation and possibly termination, are primary mechanisms regulating the rates of synthesis of some transcripts. It has been reported that transcriptional attenuation or termination is a major mechanism that regulates the rate of synthesis of mammalian mitochondrial RNA. In mammalian cells, the major heavy strand

Transcriptional Regulation of the Yeast Mitochondrial Genome
promoter is used to transcribe the genes for the tRNAPh', 12 S rRNA, tRNA""', and 16 S rRNA with little or no further transcript elongation (34-41). Thus, even though present a t the same copy number as the other genes, these genes are transcribed at much greater rate, and the rRNAs are in higher abundance (41), than the genes downstream of the 16 S rRNA.
In the case of the yeast mitochondrial genome, the regulation of the rates of transcription is determined by the strengths of the various promoters as well as by attenuation and possibly termination mechanisms. The steady state levels of the individual RNAs have been recently determined.4 These results indicate that there is a strong correlation between the relative rates of synthesis of a particular RNA and its steady state abundance. This result stresses the importance of transcriptional regulation as a primary mechanism for modulating the levels of the mtRNAs.