Proc. Nati. Acad. Sci. USAVol. 80, pp. 5564-5568, September 1983Biochemistry

Identification of a single transcriptional initiation site for theglutamic tRNA and COB genes in yeast mitochondria-

(Saccharomyces cerevwiae/guanylyltransferase/in-vitro capping/in vitro transcription/cytochrome b).

THOMAS CHRISTIANSON*, JOHN C. EDWARDSt, DAVID M. MUELLERt, AND MURRAY RABINOWIZT§Departments of tMedicine, tBiochemistry, and *Biology, The University of Chicago, Chicago, Illinois 60637

Contributed by Murray Rabinowitz, June 20,.1983

ABSTRACT We have identified a single transcriptional ini-tiation site for the glutamic tRNA and COB. (cytochrome b) genesby using the complementary techniques of in vitro capping of RNAand in vitro transcription-ln the capping reaction, mitochondrialRNA is labeled with [a-32P]GTP by vaccinia virus guanylyltrans-ferase. This reaction is specific for the 5' ends of RNA retainingthe terminal triphosphate of transcriptional initiation. Exploitingthe extremely low G+C content (18%) of yeast mitochondrial DNA,we digested in vitro capped transcripts from various petite dele-tion mutants with the G-specific RNase Ti. By petite deletionmapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNAgene. When the sequence of this oligonucleotide was determined,it perfectly matched the DNA sequence 391 bases upstream of theglutamic tRNA. Purified yeast mitochondrial RNA polymerase in-itiated transcription in vitro at the same site as shown by the se-quence of the 33-base oligonucleotide product of the reaction per-formed in the absence of CTP. Initiation starts at a nonanucleotidesequence previously implicated in yeast mitochondrial transcrip-tional initiation. Because there is no evidence of an initiation sitein the 1,050 bases between the glutamic tRNA and COB genes,the two genes are likely to be transcribed together. Further evi-dence of a long common transcript was provided by RNA blot hy-bridization.

The COB gene, coding for a subunit of the cytochrome bclcomplex of yeast mitochondria, has been the subject of inten-sive study. Production of a mature COB mRNA involves com-plex processing including splicing of several introns (1-4), theexact number of which is strain -dependent (5, 6). Some of theseintrons are unusual in that they code for maturases-that is,proteins that appear to be required for intron splicing (6-10).However, although processing mutants and intermediates in-volved in formation of mature COB message have been exten-sively analyzed, the nature of the primary transcript of this genehas not been characterized. To understand more fully the con-trol of expression of the COB gene, it is necessary to know thesite of transcriptional initiation for COB transcripts.We have recently defined.transcriptional initiation sites on

the mitochondrial genome (11-14) by using two new proce-dures. In one, vaccinia virus guanylyltransferase is used in anin vitro capping reaction to label RNA which retains its initi-ating nucleotide (11). This reaction is specific for 5'-polyphos-phate-terminated RNA molecules. By hybridization of cappedtranscripts to restriction fragments of mitochondrial DNA im-mobilized on nitrocellulose,' we previously demonstrated aminimum of four or five widely separated initiation sites (11).In addition, the exact position of the initiation sites for the largeand small ribosomal genes was determined by sequence anal-ysis of the guanylyltransferase-labeled mRNAs and matching

their sequences to known DNA sequences-11-13). In recentexperiments, we mapped 20 unique initiation sites and iden-tified the precise position on the genome for; 13 of them (15).The second procedure involves the study of. initiation with ahomologous in vitro transcription system using purified mi-tochondrial RNA polymerase and cloned DNA templates (14).We have found that the purified polymerasesis capable of spe-cific initiation -at the rRNA initiation sites identified by cap-ping.

About 25 homologous bases are found at the sites of tran-scriptional initiation for the two ribosomal genes (12, 16). Anonanucleotide conserved sequence immediately upstream fromeach rRNA initiation site is revealed by comparison of the DNAsequences in Saccharomyces cerevisiae with a distantly relatedyeast species, Kluyveromyces lactis (13). Copies of this non-anucleotide appear at a few other scattered sites in the genome(13, 17). Exact identification of initiation sites for other genesis necessary to evaluate the role of these sequences in tran-scriptional initiation.We therefore examined the initiation site for the COB gene.

The entire COB gene region, including about 3,000 bases up-stream from the translational initiation codon, has been sub-jected to sequence analysis (5, 6, 18, 19). S1 nuclease mappinghas shown that the major COB transcript extends about 900bases upstream the gene (18, 20). The next gene upstream fromCOB codes for glutamic tRNA and is separated from the COBcoding region by about 1,050 nucleotides (Fig. 1).

In this study, we identify a single transcriptional initiationsite for the glutamic tRNA and COB genes. We find that thesequence of an in vitro capped mitochondrial transcript thatmaps to this region is identical to the sequence of RNA prod-ucts transcribed in vitro from a cloned DNA template. The se-quences match the DNA sequence (19) 391 bases upstream fromthe glutamic tRNA. Initiation starts at the last base of a nonanu-cleotide sequence that is homologous-to the DNA segment pre-ceding the rRNA genes and has been implicated in promoterfunction. The glutamic tRNA and COB genes appear to sharea common primary transcript because neither nonanucleotidesequences nor other capped transcripts are observed betweenthe identified initiation site and the COB coding sequence.

MATERIALS AND METHODSStrains. Grande strains MH41-7B and D273-10B were used

in this study and have been described by Morimoto and Ra-binowitz (23). The petite strain DS400/A12 was generouslyprovided by A. Tzagoloff (Columbia University, New York). Thepetites Q3122, 01011, 0,10, and 01-2 were described by Lewinet al. (22).

Nucleic Acid Isolation. Mitochondrial RNA was prepared fromyeast cells as described (12). Mitochondrial DNA was purified

§ To whom reprint requests should be addressed.

5564

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Proc. Natl. Acad. Sci. USA 80 (1983) 5565

from similar preparations by cesium chloride density gradientcentrifugation in the presence of bisbenzimide (8). Cloned DNAwas isolated from bacterial cells by NaDodSO4 lysis and poly-ethylene glycol precipitation as described (24, 25).

Cloning:DNA. MH41-7B mitochondrial DNA. was digestedwith Hha I and separated on a 0.8% agarose gel. Hha I fragment8 was isolated and digested with Sau3A. The products were li-gated at the BamHI site of plasmid pUR222 and used to trans-form Escherichia coli strain FZ-AM15recA- as described byRuther et al. (26). All experiments with recombinant plasmidswere conducted in accordance with the National Institutes ofHealth guidelines for recombinant DNA research.

Guanylyltransferase Reactions. Guanylyltransferase wasisolated from vaccinia virions according to Levens et al. (11).The in vitro capping reactions were performed and stopped asdescribed (11, 12). The reaction mixture was extracted twicewith phenol and once with chloroform and resolved by rapidcolumn chromatography on Bio-Gel P-6 (14, 27). The excludedvolume was precipitated with 3 vol of ethanol.

In Vitro Transcription. Yeast mitochondrial RNA polymer-ase was purified as described by Levens et al. (28), except thatDEAE-Sephacel was substituted for DEAE-Sephadex and theentire purification was carried out in 0.1 mM dithiothreitol.Transcription reactions were carried out as described by Ed-wards et al. (14) for [y-32P]ATP labeling in the absence of CTP.After rapid column chromatography, the products were sepa-rated. on an 8% polyacrylamide/8.3 M urea gel.

Limit RNase TI Digestion and Extraction of Oligonucleo-tides from Gels. Mitochondrial RNA labeled at the 5' end byin vitro capping was digested to. completion with RNase Ti(Calbiochem-Behring) which cleaves specifically after G. Theseoligonucleotides were separated on polyacrylamide gels (29),excised, and extracted (12). The products of the minus CTP in

4 kb -2!

0 2 4 6 8 10 12

MH41 tRNA9'U COB OLI-I

D273 tRNA C OLI-I

sequenced DNA

DS400/A12 _Q3122

0,0

PC8- _ pLT727

FIG. 1. Genetic and physical maps of the glutamic tRNA and COBregion. Grande strain MH41 has the long version of the CORgene, andstrain D273 has the short version which lacks three introns. Open read-ing frames in the introns are stippled. Above the two grande maps isa kilobase scale, and the heavy line delineates the extent of the pub-lished DNA sequences (5, 6, 18, 19, 21). The regions of the grande ge-nome retained in the five petites-used in the deletion mappingare shownas crosshatched bars (5, 18, 22); four-of the .petites extend beyond thefigure. Deletion mapping with the petites suggests that the initiationsite (seeResults) that gives riseto the 51-base T1 oligonucleotide prod-uct is located in a 2.5-kilobase'region;this region is shown by a bracket.The two black bars at the bottom represent the mitochondrial se-quences. present in the two plasmids used in this. study; pBLT727 isaligned with the D273 map. The arrows mark the initiation-site for theglutamic tRNA and COB. genes.

vitro transcription reaction were extracted- from polyacrylamidegels as described (14).Agarose/Urea Electrophoresis and RNA Blotting. Electro-

phoresis of mitochondrial RNA was performed on 1.5% agar-ose/6 M urea gels (30). RNA was blotted onto nitrocellulose bythe procedure of Thomas (31) with the addition of a 45-minutesoak of the gels in 3 M NaCl/0.3 M sodium citrate before trans-fer.

RESULTSMitochondrial RNA from the grande strains MH41-7B and D273-lOB and from the petite strains DS400/A12, Q3122, OIOII, 0110,and OII-2 was subjected to the in vitro capping reaction with.guanylyltransferase and [a-32P]GTP. Because the G+C content*of yeast mitochondrial DNA is only 18%, primary transcriptsvary greatly in the number of bases from the initiating nucleo-tide to. their first G. By exploiting this feature, the capped mi--tochondrial RNAs from each strain were completely digestedwith the G-specific RNase T1. Afterward, the oligonucleotideproducts were subjected to electrophoresis and autoradiogra-phy. An example of this type of digest of capped RNA from thepetite 0110 is presented in Fig. 2.

A.LG

-50- 05140 -

20-

10--

10--000

dw

B.G -C IPy G Pyi A,:\~~~~~~~~~-

_*--A

_o -UA--d * - u

*- A

FIG. 2. Analysis of the 51-base oligonucleotide. (A) In lane G, mi-tochondrialRNA from petite Od10 was capped in vitro, digested to com-pletion with the G-specific RNase T1, and electrophoresed on a 20%polyacrylamide sequencing gel. The T1 products are sized by compar-ison to the-adjacent lane L, a reference ladder made by partial alkalinedigestion (29) oftotal cappedRNA. Two oligonucleotides are visualized,one of 11 bases and another of approximately 51 bases. Several addi-tional faint oligonucleotides appeared after longer exposure ofthe com-plete T1 digest of petite Oi10. (B) The 51-base oligonucleotide was iso-lated and its-sequence was determined by partial digestion with base-specific RNases. Conditionsfor all enzymatic digestions were essen-tially as described by Silberklang et al. (32), except that the U2 diges-tion was performed at pH 3.5. The lanes of the 20% polyacrylamide se-.quencing gel (29) were: A, RNA digested with the A-specific RNase U2;Py, with the U- and C-specific RNase from Bacillus cereus; -C, withthe G-, A-, U-specific RNase Phy I; G, with the G-specific RNase T1.

Biochemistry: Christianson et al.

5566 Biochemistry: Christianson et al.

We observed a number of unique capped oligonucleotides inthe digests from both grande strains and from four of the petitestrains but not in the small (5-kilobase) petite 011-2. Becausethe petites retain different regions of the mitochondrial ge-nome (Fig. 1), the transcriptional initiation sites can be local-ized by deletion mapping. We have mapped a 50-base oligo-nucleotide to a 2.5-kilobase region upstream of the glutamictRNA (Fig. 1). The oligonucleotide was present in the digestsfrom petites Q3122, 01011, and 0110 and also was observed inRNA from the grande strains (data not shown). The transcriptfrom which the 50-base oligonucleotide arises must originate inthe region of the overlap between petites Q3122 and 0101I.The absence of the 50-base oligonucleotide from limit T1 di-gests of capped RNA from 011-2 and DS400/A12 further sug-gests that the transcript originates within the 2.5-kilobase re-gion indicated in Fig. 1.The oligonucleotides arising from the petites were excised

from polyacrylamide gels, eluted, and subjected to enzymaticsequence analysis reactions. The approximately 50-base oli-gonucleotides from petites Q3122, 01011, and 0110 proved tohave the same sequence. On a 20% polyacrylamide gel, the se-quence could be read clearly from the 1st to about the 36th base(Fig. 2). A perfect match for the 36 bases was found in DNAsequenced by Goursot et al. (19), starting 391 bases upstreamof the glutamic tRNA gene. The first G in the transcript is 51bases downstream from the initiation site, establishing the ex-

A. B.T

-U,.

....Um

75 - 31

O.-* -- Al- U

- A-AA

- AA

_ -U_W -A

_ -U

0 -A

37 -

e -33

act length of the capped T1 oligonucleotide.An imperfect nonanucleotide initiation sequence (in which

G replaces an A at position -3), previously noted by Baldacciand Bernardi (17), was at the initiation site of the 51-base oli-gonucleotide. Transcription started at the last A of the nine-basesequence, which is also the case for the initiation sites of theribosomal genes (11-13). No other copies of the nonanucleotideinitiation sequence are present in the DNA downstream fromthis site to the glutamic tRNA gene, nor are copies present inthe 1,050 bases of DNA between the tRNA and COB genes.Furthermore, none of the sequences of other capped oligo-nucleotides derived from petite or grande mitochondrial RNAare found in this region (15). These results indicate that the glu-tamic tRNA and COB genes are cotranscribed as a multigeneprecursor from the same initiation site.

The second approach to study of the initiation of the glu-tamic tRNA and COB genes was by in vitro transcription. Wepreviously showed that purified yeast mitochondrial RNA poly-merase initiates transcription in vitro with high fidelity at therRNA genes (14). In order to study the glutamic tRNA tran-scriptional initiation site with this system, a 700-nucleotide Sau3Afragment containing the initiation site was cloned into the BamHIsite in the vector pUR222 (26). This clone was designated pC8,and the region it contains is shown in Fig. 1. RNA was syn-thesized from the supercoiled pC8 template by using purifiedmitochondrial RNA polymerase with [y-32P]ATP in the absenceof CTP. Correct initiation would be expected to yield a tran-script, uniquely labeled at its 5' initiating end, that terminatesat the first C (position 34) in the transcript.

Polyacrylamide gel electrophoresis of the products of in vi-tro transcription from pC8 in the absence of CTP reveals thatthe major product has approximately the expected size of 33nucleotides (Fig. 3). To confirm the identity of this transcript,the 33-nucleotide band was purified from the gel and its se-quence was determined (Fig. 3). The sequence was the sameas that obtained for the capped 51-base oligonucleotide. Thecomplementary results from the two techniques further prove

10000-

5000-4.

UL 2000-

°i 1000

z 500-

2004

- u

-A

19 a

a

A B C

(DNA)- '(b

.421 S- b _

(28 )-" I

iss1S-6-(18S) 0

FIG. 3. Analysis of the 33-base oligonucleotide transcribed in vitroin the absence of CTP. (A) In lane.T, the products of in vitro transcrip-tion.in the absence ofCTP were separated on an8% polyacrylamide gel.The products were sized by reference to labeled markerDNA fragmentsin lane M. An oligonucleotide of about 33 bases was visualized. (B) The33-base oligonucleotide was isolated and analyzed by partial digestionwith base-specific RNases. The code used for the lanes is as in Fig. 2,except that U refers to the digest with the pyrimidine-specific RNasefrom B. cereus.

FIG. 4. RNA blot hybridizations. Lanes: A, ethidium bromide flu-orescence pattern of MH41 mitochondrial RNA separated on a 1.5%agarose/6 M urea gel; B, nitrocellulose blot of A probed with the 700-.base Sau3A fragment carrying the glutamic tRNA initiation site iso-lated from clone pC8; C, nitrocellulose blot ofA probed with the COB-specific clone pBLT727. A high molecular weight transcript common toboth hybridizations is marked with an arrow. The labels for the con-taminating cytoplasmic 28S and 18S RNA species are enclosed in pa-rentheses. Hybridizations wereperformed as described (12), except thatDNA from petites F11 and P9, which do not contain the COB region(22), were used as carrier to decrease possible cross hybridization.

Proc. Natl. Acad. Sci. USA 80 (1983)

Proc. Natl. Acad. Sci. USA 80 (1983) 5567

4iwfioting nuceofide-/0 ATATAAGT A /0 20 30 40 50

DM4 ATA'TTATATAGGT AATATATAAA'AATAATATAA'AATAATTATAATTCAATTTATATATTAATAMTTCC-*RM AAUAUAUAAAAAUAAUAUAAAAUAAUUAUAAUUCAA- - -6

FIG. 5. The glutamic tRNA initiation site. The sequence at the 5' end of the glutamic tRNA-COB transcript, as determined from the in vitrocapped oligonucleotide, is compared to the homologous strand of the DNA (21). The initiation site is marked with an arrow. Above the two sequencesis the consensus nonanucleotide initiation sequence; note that in the DNA sequence there is a single base alteration from the consensus at position-3.

the precision of the in vitro transcription system and ensurethat both techniques are reliable probes for transcriptional ini-tiation.

Because neither the nonanucleotide initiation sequence northe sequences of any in vitro capped transcript can be foundbetween the initiation site we have identified and the COB gene,it is likely that the glutamic tRNA and the COB genes are tran-scribed as a common transcript from this initiation site. Wetherefore sought to identify such a common precursor in grandemitochondrial RNA by blot hybridization. MH41 mitochondrialRNA was electrophoresed on agarose/urea gels and blotted ontonitrocellulose. Identical strips were hybridized with probesspecific either for the initiation site (pC8) or for the 3' end ofthe COB coding region (cloned EcoRI fragment 6, pBLT727)(33). These hybridizations were visualized by autoradiography(Fig. 4). A number of transcripts are seen, especially with theCOB-specific probe. A long transcript is seen to be common toboth probes. This transcript is at least 9,000 nucleotides long,adequate to contain the initiation site and the 3' end of the COBgene (long version of the gene). Conclusions based on blot hy-bridization must be treated with some caution because crosshybridization has been observed with yeast mitochondrial DNAprobes (34). However, the data support the view that a commontranscript derived from the glutamic tRNA and COB genes ispresent in the mitochondria.

DISCUSSIONWith the complementary techniques of in vitro capping oftranscripts isolated from mitochondria and in vitro transcrip-tion with purified RNA polymerase, we have found a tran-scriptional initiation site preceding the glutamic tRNA gene.Because no other capped transcripts nor the nonanucleotideconsensus sequence could be found between the glutamic tRNAgene and the COB gene, these genes apparently share a com-mon initiation site and transcript. This primary transcript ex-tends 391 nucleotides upstream from the tRNA gene. Becauseby RNA blot hybridization we find a low abundance of putativeprecursor which hybridizes to probes from the COB coding re-gion and from the region of the initiation site, the expected pri-mary transcript appears to be present in yeast mitochondria.

There have been several other reports of multigene tran-scripts in yeast mitochondria. At least one tRNA gene is in-cluded on the 3' end of the precursors of the 21S rRNA gene(35, 36), and multigene tRNA transcripts have also been dem-onstrated (37). Like the common transcript for the glutamic tRNAand COB genes, a tRNA (valine tRNA) and protein gene (OXI-2) have been shown to be transcribed together (38).

Although multigene transcripts are present in yeast mito-chondria, the yeast mitochondrial DNA is not transcribed intogenome-length transcripts. In this regard, yeast mitochondriadiffer from animal mitochondria. In addition to the separatetranscriptional initiation sites for the ribosomal genes (11-13),we have evidence for more than 20 other initiation sites (11,15). The existence of multiple transcriptional units suggests thata high degree of coordination and regulation is involved in theexpression of the yeast mitochondrial genome.The conserved nonanucleotide initiation sequence is likely

to be necessary for transcription in yeast mitochondria becauseit occurs not only at the ribosomal genes and the glutamic tRNAbut also at each of 10 other identified origins of transcription(15). In the case of the glutamic tRNA-COB transcriptional unit,the nonanucleotide sequence is imperfect, with a G replacingan A at position -3 from the initiating nucleotide (Fig. 5). Thisindicates that the nonanucleotide initiation sequence need notbe perfectly conserved. The single base substitution in thenonanucleotide initiation sequence may be involved in mod-ulating rates of transcription because the steady-state level ofcappable ends from the glutamic tRNA-COB initiation site ismuch lower than the level of cappable ends from the rRNAs.The common presence of the nonanucleotide initiation se-

quence before all the demonstrated transcriptional units andthe existence of multigenic transcripts with tRNA genes andprotein or rRNA genes show that the same RNA polymeraseis used for all mitochondrial transcripts. This conclusion isstrengthened by the fact that the same purified RNA poly-merase transcribes the ribosomal RNA and the glutamic tRNA-COB genes. Both the capping reaction and the results from invitro transcription show that the polymerase is highly specificfor the transcriptional initiation sites.

We thank Drs. Lucia Rothman-Denes and Joseph Locker for theircritical review of the manuscript. This study was supported in part byNational Institutes of Health Grants HL-04442 an HL-09172, by GrantNP-281 from the American Cancer Society, and by a grant from theLouis Block Fund of the University of Chicago. J.C.E. and D.M.M.were supported by training grants from the National Institutes of Health.

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