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Volume 271, Number 11, Issue of March 15, 1996 pp. 6537-6544
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Characterization of the Neurospora crassa cyt-5 Gene
A NUCLEAR-CODED MITOCHONDRIAL RNA POLYMERASE WITH A POLYGLUTAMINE REPEAT (*)

(Received for publication, November 16, 1995)

Bing Chen (1),  Anne R. Kubelik (§),  ,  Sabine Mohr ,  Caroline A. Breitenberger (¶)

From the Departments of Biochemistry and Molecular Genetics and the Ohio State Biochemistry Program, Ohio State University, Columbus, Ohio 43210-1292

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Neurospora crassa mutants, cyt-5-1 and cyt-5-4, have a cytochrome b- and aa(3)-deficient phenotype, suggesting that they result from a deficiency in a nuclear-coded component of the mitochondrial gene expression apparatus (Bertrand, H., Nargang, F. E., Collins, R. A., and Zagozeski, C. A.(1977) Mol. Gen. Genet. 153, 247-257). The complementing wild-type gene has been cloned and sequenced, and shown to encode a protein with significant sequence similarity to Saccharomyces cerevisiae mitochondrial RNA polymerase and bacteriophage RNA polymerases. There are remarkable differences between the N. crassa protein and its yeast homologue, including a region of very little homology near the N termini of the two gene products. The cyt-5 gene encodes a stretch of polyglutamine in this region of unique sequence. In addition, an acidic insertion (86 amino acids, of which 24 are Asp or Glu and 10 are Arg or Lys) is present near the C terminus of the cyt-5 gene product. Transcript levels of the cytochrome b and cytochrome oxidase subunit III genes are severely reduced in cyt-5 mutants, suggesting a likely mechanism for the cytochrome-deficient phenotype. In contrast, mitochondrial rRNAs accumulate to nearly normal levels in cyt-5 mutants. However, mitochondrial rRNA levels are not indicative of the rate of transcription of the corresponding genes, since crude lysates of mitochondria from cyt-5 mutants exhibit greatly reduced transcriptional activity with a 19 S rRNA promoter. The cyt-5 gene is flanked by at least one gene whose product also may be involved in mitochondrial function.

INTRODUCTION

A distinct RNA polymerase is required in eukaryotic cells solely for the purpose of transcribing genes encoded by the mitochondrial genome. To date, the best characterized mitochondrial RNA (mtRNA) polymerase is that of Saccharomyces cerevisiae(1, 2) . S. cerevisiae mtRNA polymerase consists of a 145-kDa core polymerase and a specificity factor. The core polymerase as well as the known specificity factors are encoded by nuclear genes(3, 4, 5) . The core polymerase displays strong sequence similarity to bacteriophage RNA polymerases(6) , which do not require specificity factors for promoter recognition(7) . Although no other mtRNA polymerase sequences have been determined, it is likely that the similarity of mtRNA polymerases to bacteriophage RNA polymerases is not unique to yeast, as the purified Xenopus laevis mtRNA polymerase also consists of a 140-kDa core subunit with an associated specificity factor(8) .

Despite extensive biochemical characterization of the Neurospora crassa mitochondrial RNA polymerase(9, 10) , the corresponding gene has not yet been analyzed. However, Bertrand (11) and Bertrand et al.(12) have described a nuclear mutant of N. crassa, cyt-5-4, which has the characteristics expected of a mutation in a nuclear gene involved in mitochondrial gene expression. This slow-growing mutant is deficient in cytochromes b and aa(3), which are wholly or partially encoded by mitochondrial genes, while levels of the nuclear-coded cytochrome c are elevated. We investigated how this mutant and another allele, cyt-5-1, affect mitochondrial gene expression.

We report the cloning and sequencing of the wild-type cyt-5 gene of N. crassa, which encodes a product homologous to yeast mtRNA polymerase as well as bacteriophage RNA polymerases. Phenotypic analysis of the two cyt-5 mutants as well as amino acid sequence comparisons suggest that the cyt-5 gene product corresponds to the N. crassa mitochondrial RNA polymerase. Differences in amino acid sequence between the N. crassa and yeast mtRNA polymerases are discussed.

MATERIALS AND METHODS

Strains of Neurospora and Growth Conditions

The wild-type N. crassa strain was 74-OR23-1A; FGSC 2489 and the poky strain was [poky] (13-6A) (13) . The mutant strains were cyt-5-1A pan-2 (1061 EA76A) and cyt-5-4a pan-2 (1062 HM50a)(12) . Both strains were a gift of H. Bertrand, University of Guelph. The strain cyt-5-1A inl (C5Q152) was derived from a cross between cyt-5-1A pan-2 and ARK-1a qa-2 aro-9 inl and used for transformation. Procedures for maintaining strains, carrying out genetic crosses, and growing conidia and mycelia were as described(14, 15) . The growth times for mycelia in liquid culture were the following: 74A: 14 h at 25 °C or 10 h at 37 °C; cyt-5-1 and cyt-5-4 strains: 24 h at 25 °C or 36-48 h at 37 °C; cyt-5-1 transformants: 12 h at 37 °C.

Cytochrome Spectra

Reduced minus oxidized difference spectra of crude mitochondria were obtained as described(16) , using a Beckman DU-7 spectrophotometer.

Cloning by Complementation

The cyt-5 gene was cloned using sib selection essentially as described(17) , from the cosmid library of Vollmer and Yanofsky(18) , which consists of N. crassa chromosomal fragments from the laboratory wild-type 74A cloned into pSV-50, a pBR322-derived vector containing the cos site and the N. crassa benomyl-resistance (bml) gene. The sib selection procedure of Akins and Lambowitz (17) involves transforming the mutant strain with pools of DNA (sibs) of successively decreasing complexity. cyt-5-1A inl was made competent for transformation after 6 h germination, using 15 mg/ml Novozym 234 according to Akins and Lambowitz(17) . For the first round of transformation, 5 µg of CsCl-purified DNA and 2 times 10^8 cells in a final volume of 160 µl were used. For all subsequent rounds, approximately 1 µg of DNA prepared by the potassium acetate method (17) and 5 times 10^7 cells in a final volume of 40 µl were used. Following transformation, cells were suspended and plated as described(19) . Transformants were allowed to regenerate at 25 °C for 4-12 h under nonselective conditions before benomyl diffused in from the bottom agar. The plates were then transferred to 40 °C, because, although cyt-5-1 grows slowly at all temperatures, the background of untransformed colonies was lowest if the transformants were grown at the higher temperature. One plate was left at 25 °C to monitor the efficiency of bml selection. After 1 day at 40 °C, plates were checked for colonies, which were picked onto slants and placed at 40 °C. Strains that grew rapidly, with conidiation, were considered to be stable transformants. The final clone, obtained after four rounds of sib selection, was called pSV50-cyt-5.

pSV50-cyt-5 was digested with restriction enzymes and individual restriction fragments were purified by agarose gel electrophoresis. The cyt-5-1A inl mutant was co-transformed with gel-purified fragments and plasmid pSV-50 to provide the intact bml gene(20) . A 9.0-kb (^1)HindIII fragment with full transforming activity was subcloned into the vector pBS(+) (Stratagene) to construct plasmid C5H2-26. The transforming activity was further localized by transforming cyt-5-1 with gel-purified fragments of C5H2-26 and pSV-50 as described above.

DNA Sequence Analysis

Overlapping 2.8-kb EcoRI and 3.2-kb BglII fragments of C5H2-26 were subcloned into the EcoRI and BamHI sites, respectively, of vector pGEM4Z (Promega Biotech Inc.). Deletions were generated from both ends of these clones using the Erase-A-Base System (Promega Biotech) and the sequence of both strands was determined. Plasmid DNAs, prepared by the alkaline method (21) were sequenced by the dideoxy method using universal and reverse sequencing primers according to the Sequenase kit from U. S. Biochemical Corp.

The DNAStar package of computer programs was used for general DNA and amino acid sequence analysis. Sequence comparisons relied on the Blast E-mail server at the National Center for Biotechnology Information at the National Library of Medicine(22) .

RNA Preparation

Total RNA was prepared from frozen mycelial pads by a modification of the procedure of Hoge et al.(23) . Briefly, the mycelial pad was ground to a powder in a prechilled mortar and then quickly mixed with 10 ml of preheated lysis buffer (lysis buffer: 0.2 M boric acid, 30 mM EDTA, 1% SDS, pH adjusted to 9.0 with NaOH) and 10 ml of buffer-saturated phenol/chloroform/isoamyl alcohol (25:24:1) at 55 °C. After thorough mixing, the phases were separated by centrifugation, and the aqueous layer was transferred to a new tube and extracted with phenol three times more. To the final supernatant were added one-tenth volume of 3 M sodium acetate and 2-3 volumes of ethanol. The RNA was pelleted and washed twice with 70% (v/v) ethanol. The nucleic acid pellet was resuspended in diethylpyrocarbonate-treated water and 8 M LiCl solution was added to a final concentration of 2 M. The mixture was incubated at least 2 h on ice, at which point the RNA was pelleted by centrifugation. The pellet was washed twice with 70% ethanol and resuspended in RNase-free water. Aliquots of the RNA were stored as ethanol precipitates at -70 °C.

Mitochondria were isolated by the modified flotation gradient method of Lambowitz(15) . Total nucleic acids were isolated from mitochondria by the same procedure described for whole cell RNA, except that the weight of mitochondrial pellet was determined accurately and the LiCl precipitation step was omitted.

Northern Blots

RNAs (20 µg for each lane) were separated by electrophoresis on 1% formaldehyde-agarose gels(21) . Gels were stained, photographed, and transferred to Hybond-N (Amersham) by capillary transfer in 20 times SSC(21) . The blots were probed with various labeled DNA fragments in hybridization solution containing 5 times SSC, 5 times Denhardt's solution, 50% formamide, 0.1% SDS, and 10 µg/ml denatured salmon sperm DNA at 42 °C. Prehybridization was carried out in the same solution for at least 2 h at 42 °C before addition of labeled probes. After overnight hybridization, the blots were washed sequentially with 1 times SSC, 0.1% SDS at room temperature for 15 min twice, 0.5 times SSC, 0.1% SDS at 65 °C for 15 min twice, and if necessary, additional washes were carried out with 0.1 times SSC, 0.1% SDS at 65 °C for 15 min once or twice. The various probes used for Northern blots were the recombinant plasmids pAPHc660 (24) containing a DNA fragment from the mitochondrial 19 S rRNA gene, pHX9422 (25) for the 25 S rRNA gene, pLB3 with a fragment from the cob gene, (^2)pAP11 (24) for the coIII gene, and pBen (26) for the beta-tubulin gene. Probes were labeled by the random primer labeling method with [alpha-P]dCTP(21) .

For the normalization of blots containing total RNA, one or more trial gels were run, with varying amounts of RNA loaded in each lane. The blots were transferred to Hybond-N as above and probed with a beta-tubulin probe. For the experimental gels, the amount of RNA in each lane was adjusted to yield equivalent amounts of beta-tubulin mRNA, as determined by the intensity of the corresponding band on the trial gel.

RNA Polymerase Assay

Run-off transcription assays were carried out as described by Kennell et al.(10) . Briefly, mitochondria were prepared by the flotation gradient method as described (15) except that the final pellet was washed with HKMTD solution containing 25 mM Tris-HCl (pH 7.5), 0.3 M KCl, 15 mM MgCl(2), and 5 mM dithiothreitol. Frozen mitochondrial pellets were lysed in HKMTD containing 1% Nonidet P-40 and the lysates were obtained by centrifugation at 12,500 rpm with JA-17 rotor (Beckman) for 15 min. The protein concentration of the lysates was determined by a Coomassie-binding assay (Bio-Rad). The DNA template was EcoRV-digested plasmid pSRBP-1, which contains the promotor of the mitochondrial small (19 S) rRNA, yielding an expected 325-nucleotide run-off transcript(10) . Lysates were usually preincubated with antiserum against the Neurospora endo/exonuclease (a gift from T. Chow, Université de Montréal) on ice for 30 min. The transcription assays were carried out in 20 µl of reaction medium containing 2 µl of lysate (1 µg of protein), 2 µl of antiserum concentrated by centrifugation through a Centricon-10 filter (Amicon), 200 ng of DNA template, 10 mM Tris-HCl (pH 8.0), 10 mM MgCl(2), 20% glycerol, 0.1 mg/ml acetylated bovine serum albumin (New England Biolabs), 150 µM ATP, GTP, and CTP, 5 µM UTP (Pharmacia), and 20 µCi of [alpha-P]UTP (3,000 Ci/mmol, DuPont NEN). Transcription was initiated by the addition of template and nucleotides, and allowed to proceed for 10 min at 30 °C, followed by addition of 1 µl of 1 mM UTP and an additional 10 min at 30 °C. The reactions were terminated by extraction with phenol/chloroform/isoamyl alcohol (25:24:1), followed by ethanol precipitation. Transcription products were analyzed by electrophoresis on 8% polyacrylamide-urea gels followed by autoradiography.

RESULTS

Cyt-5 Mutants Exhibit a Cytochrome-deficient Phenotype

The N. crassa nuclear mutant, cyt-5-4, has been described previously to have a slow-growing, cytochrome deficient phenotype(12) . The absence of cytochromes b and aa(3) in the allelic cyt-5-1 mutant is evident, based on the loss of peaks at 560 and 605 nm, in reduced minus oxidized cytochrome spectra (Fig. 1b). Cytochrome b and subunits I, II, and III of cytochrome aa(3) (cytochrome oxidase) are encoded by genes located on mitochondrial DNA. Cytochrome c, which is encoded by a nuclear gene, is somewhat over-expressed in the cyt-5-1 and cyt-5-4 mutants (12, Fig. 1b). The observed deficiency in all cytochromes with mitochondrially synthesized subunits suggests that the cyt-5 gene product is essential for mitochondrial gene expression.

Figure 1: Cytochrome spectra of cyt-5 mutants. Difference spectra (reduced minus oxidized) of crude mitochondria from wild-type 74A (a), cyt-5-1 (b), and two normally growing isolates of cyt-5-1 transformed with pSV50-cyt-5 (c and d) are shown. The peaks at 550, 560, and 605 nm are the alpha-peaks of cytochromes c, b, and aa(3), respectively.


Cloning and Complementation of the cyt-5Gene

The cyt-5 gene was cloned from a cosmid library by complementation of the cyt-5-1 mutant using the sib selection procedure of Akins and Lambowitz(17) . A double selection scheme was employed in the cloning of the cyt-5 gene. Transformants were grown in the presence of benomyl to select for the bml marker supplied by the pSV-50 cosmid vector. Selection for cyt-5 involved growing transformants at 40 °C, where untransformed cyt-5-1 colonies grow poorly. After four rounds of sib selection, the cosmid pSV50-cyt-5, with an insert size of approximately 40 kb, was isolated. Cytochrome spectra of cyt-5-1 transformants with pSV50-cyt-5 are similar to those of wild-type N. crassa mitochondria (Fig. 1, c and d) with peaks at 560 and 605 nm, indicating the presence of cytochromes b and aa3.

The genomic location of the DNA fragment cloned by sib selection was determined by restriction fragment length polymorphism mapping(27) , using the restriction enzyme HindIII (not shown). The pSV50-cyt-5 insert hybridized to polymorphic restriction fragments showing linkage to 5 S rRNA genes 62 and 63 (17/18 progeny) and to cot-1 (15/18 progeny). These mapping data agree with genetic data showing that cyt-5 is located 5.3 map units from arg-2 and 2.2 map units from arg-14 on linkage group IV(28) .

The cyt-5 transforming activity was localized within pSV50-cyt-5 by cotransformation of cyt-5-1 with gel-purified restriction fragments of the insert in the pSV50 clone to provide the cyt-5 gene and with intact pSV-50 to provide the bml selectable marker. Transformants were scored, as before, by growth at 40 °C in the presence of benomyl. Full transforming activity was localized to a 9.0-kb HindIII fragment (Fig. 2). This fragment was subcloned, and the cyt-5 gene was further localized by analyzing the transforming ability of gel-purified restriction fragments of the HindIII subclone (Fig. 2).

Figure 2: Cloning and complementation of the cyt-5 gene. A restriction map of the cyt-5 gene is shown, with the following abbreviations for restriction enzyme sites: B, BglII; EI, EcoRI; EV, EcoRV; H, HindIII; Hp, HpaI; N, NruI; S, SphI; and Sa, SalI. The cyt-5 open reading frame and its orientation are indicated by the arrow above the map. The location of the stretch of polyglutamine and the acidic insertion discussed in the text are indicated by ``Q'' and ``///,'' respectively. Restriction fragments used to complement the cyt-5-1 mutant are indicated below the map, with cyt-5 complementation indicated by +.


DNA Sequence Analysis Suggests That the cyt-5Gene Encodes a Mitochondrial RNA Polymerase

The sequences of overlapping 2.8-kb EcoRI and 3.2-kb BglII subclones of the 9.0-kb HindIII fragment were determined, revealing a continuous open reading frame of 1422 amino acids (indicated by the arrow, top of Fig. 2). Following the first ATG codon is a sequence rich in hydroxylated amino acids (Ser and Thr) containing regularly spaced basic residues, consistent with the general characteristics of mitochondrial transit peptides(29) . No sequence matching N. crassa intron consensus sequences (30) could be identified within or near the putative open reading frame, which encodes a polypeptide of 156 kDa.

A computer search of the GenBank sequence data base revealed that the cyt-5-encoded open reading frame has strong homology with the nuclear-coded mitochondrial RNA polymerase of S. cerevisiae(6) and to bacteriophage T3, T7, and SP6 RNA polymerases(31, 32, 33) . An alignment of the cyt-5 open reading frame with S. cerevisiae mitochondrial RNA polymerase and with T7 bacteriophage RNA polymerase is shown in Fig. 3. Homology of the cyt-5 gene is generally stronger to yeast mitochondrial RNA polymerase than to the bacteriophage RNA polymerase. However, there is a region near the N terminus of the cyt-5 protein (the first 350 or so amino acid residues) with at best limited sequence similarity to the yeast gene product. Within this domain is a sequence of 19 glutamines interrupted by one glutamic acid (residues 278-297). The S. cerevisiae mitochondrial RNA polymerase gene does not share this polyglutamine sequence. Near the C terminus of the cyt-5 gene is an 86-amino acid insertion, relative to yeast mitochondrial RNA polymerase, that is relatively rich in charged amino acids, especially acidic ones (residues 1289-1374). The relative locations of the polyglutamine and acidic region are indicated above the complementation data in Fig. 2.

Figure 3: Alignment of the cyt-5 amino acid sequence (derived from the DNA sequence) with RNA polymerases of S. cerevisiae mitochondria (6) and T7 bacteriophage (32) . Identical amino acids are indicated by (), and (bullet) indicates similar amino acids between two sequences. Gaps introduced to increase similarity between the sequences are indicated by hyphens. Amino acids are numbered at the end of each line. Asterisks mark amino acid residues conserved in all of the primitive DNA-dependent RNA and DNA polymerases(39) . Restriction sites indicated in Fig. 3are shown above the cyt-5 sequence. The location of the stretch of polyglutamine and the acidic insertion discussed in the text are indicated by Q and ///, respectively.


Expression of the cyt-5 Gene

The poky mutant of N. crassa has a deficiency in small ribosomal subunits due to a 4-base pair deletion in the mitochondrial 19 S rRNA promoter(13) . The consequent decrease in 19 S rRNA levels in poky mitochondria results in a deficiency in small ribosomal subunits and impaired mitochondrial protein synthesis, which, in turn, result in a slow-growing (hence ``poky'') phenotype. Many nuclear genes encoding components of the mitochondrial genetic apparatus are at least 5-fold over-expressed in poky(34) . This response seems to be specific to genes required for mitochondrial function: beta-tubulin mRNA, for example, is not over-expressed in the poky background. (^3)

The cyt-5 gene is also over-expressed in the poky mutant of N. crassa, suggesting that its gene product is involved in mitochondrial function. DNA fragments containing the cyt-5 open reading frame, including the 2.8-kb EcoRI fragment, hybridize to a 5.6-kb RNA that is more abundant in poky than in wild-type ( Fig. 4and Fig. 5). N. crassa mitochondrial RNA polymerase activity was reported to be higher in poky than wild-type mitochondria(10) , and was similarly induced by treatment with antibiotic inhibitors of mitochondrial protein synthesis(35) .

Figure 4: Transcription analysis of the cyt-5 gene. RNA blots containing total RNA from wild-type (left) and poky (right) were normalized based on beta-tubulin mRNA levels and probed with the probes (A-E) indicated at the bottom. Sizes of hybridization products were determined by comparison with marker RNAs (BRL, 0.24-9.5 kb RNA marker) on the same gels (not shown).


Figure 5: Transcription of the cyt-5 gene in cyt-5 mutants. A blot of total RNA (20 µg) from cyt-5-4 (lane 1), cyt-5-1 (lane 2), and poky (lane 3) mutants is compared with wild-type (lane 4). RNAs were normalized by amounts of beta-tubulin transcript. The probe was the 9-kb HindIII fragment containing the entire cyt-5 gene and flanking sequences. The 5.6-kb transcript is indicated by the arrow at right.


The approximate position of the 5`-end of the cyt-5 transcript was mapped by Northern hybridization using probes extending from the upstream EcoRI site to nucleotide -290, -100, or +310 (where +1 is the presumptive initiation codon of the cyt-5 gene). All of these probes hybridize to the 5.6-kb poky-induced transcript (Fig. 4, B, C, and D), indicating that the 5`-end of cyt-5 mRNA is at least 260 base pairs upstream of the presumptive ATG initiation codon (assuming at least 30 complementary nucleotides are required for detectable hybridization). A shorter probe, extending from the EcoRI site to nucleotide -510, no longer hybridizes to this transcript (Fig. 4A), suggesting that most or all of this probe lies upstream of the 5`-end of the cyt-5 transcript. Taken together, the results shown in Fig. 4indicate that cyt-5 transcription initiation occurs within the interval 260 to 540 nucleotides upstream from the presumptive initiation codon. Although termination codons are found in all reading frames, there are no ATG codons within this interval of the cyt-5 gene sequence: the closest ATG codon is 620 base pairs upstream from the presumptive initiation codon, and is therefore not present in the 5`-untranslated region of the mRNA.

To confirm that the cyt-5 transcript does not extend further upstream than the EcoRI site, the 1-kb HindIII-EcoRI fragment upstream from the cyt-5 gene was subcloned and used as hybridization probe against wild-type and poky total RNA, as shown in Fig. 4E. Interestingly, although no transcript corresponding to cyt-5 was seen, a smaller transcript hybridized to the upstream fragment, suggesting that a second gene lies immediately upstream from the cyt-5 gene. Furthermore, the transcript of this upstream gene is also induced in poky. Based on the observation that this gene is coordinately regulated with cyt-5 in response to a mitochondrial deficiency, we surmise that its product is also involved in mitochondrial function.

To determine whether the cyt-5 gene is transcribed in cyt-5-1 and cyt-5-4 mutants, total RNA was prepared from these mutants and hybridized with a cyt-5 probe (Fig. 5). The cyt-5-4 mutant appears to produce similar amounts of cyt-5 gene transcript compared to the wild type. However, the cyt-5-1 mutant fails to produce normal amounts of the corresponding transcript, suggesting that this mutant may be defective in cyt-5 transcription or mRNA stability. A longer exposure of lane 2 of Fig. 5does reveal low levels of cyt-5 transcript in the cyt-5-1 mutant. The normal levels of transcript in the cyt-5-4 mutant suggest that the defect in this mutant lies in post-transcriptional expression or in the gene product itself.

Mitochondrial RNA Levels in cyt-5 Mutants

Hybridization probes corresponding to mitochondrial rRNA genes were used to determine whether the observed deficiency in cytochromes b and aa(3) in cyt-5 mutants could be due to a gross abnormality in rRNA levels or RNA processing. Normalization of RNA blots based on mtDNA levels might not be reliable since mutations in yeast mitochondrial RNA polymerase decrease mtDNA levels, presumably by affecting priming for DNA replication(36) . Furthermore, mitochondrial RNA and protein levels could not be used for normalization because the cyt-5 gene potentially encodes a mtRNA polymerase. To overcome this problem, levels of rRNAs in cyt-5 mutants were established by analyzing mitochondrial and total RNA, and by using different methods for normalizing amounts of RNA loaded per lane. In Fig. 6A, 1, the amount of total RNA in each lane was normalized by hybridization with N. crassa beta-tubulin, a constitutively expressed nuclear-coded gene (26) . In Fig. 6A, 2 and 3, mitochondria were isolated prior to RNA extraction, and RNA levels in each lane were normalized based on the starting wet weight of mitochondria. Blots were hybridized with probes corresponding to mitochondrial rRNAs (19 S and 25 S rRNA). Regardless of the starting material for the RNA preparation, or how RNA levels were normalized, the results are in agreement: rRNA levels in cyt-5 mutants are only slightly lower than wild-type. No differences were observed in the relative levels of 19 S and 25 S rRNAs. There are no obvious RNA processing defects in the rRNAs detected in cyt-5 mutants. Specifically, the group I intron in 25 S rRNA apparently splices normally.

Figure 6: Comparison of rRNA and mRNA transcript levels in cyt-5 mutants and wild type. A, mitochondrial rRNA levels in cyt-5 mutants are almost normal compared to wild-type N. crassa. 1, total cellular RNA (20 µg), from the wild-type, cyt-5-1, and cyt-5-4 mutants, was subjected to formaldehyde-agarose gel electrophoresis and probed with the 19 S rRNA probe. RNA loading was normalized based on beta-tubulin mRNA levels. 2, mitochondrial RNA from wild-type, cyt-5-1, and cyt-5-4 mutants was probed with the 19 S rRNA probe. The amount of RNA loaded in each lane corresponds to 2 mg of starting wet weight of the isolated mitochondrial pellet. 3, as in 2, except that the probe was a 25 S rRNA gene fragment. The slight difference in electrophoretic mobility in lanes containing mutant mitochondrial RNA is probably an artifact of the reduced levels (overall) of RNA in the cyt-5 mutants, and is observed with probes for other RNAs also (e.g. B, 2). B, mitochondrial RNA normalized according to the wet weight of pelleted mitochondria was hybridized with a cloned fragment of the gene for cytochrome oxidase subunit III. Different lanes are: wild-type mitochondrial RNA, mitochondrial RNA from the cyt-5-1 mutant, mitochondrial RNA from the cyt-5-4 mutant. 2, as in 1, except that the probe corresponds to the cytochrome b gene. Sizes of hybridization products were determined by comparison with marker RNAs.


In contrast to the rRNA probes, the results of Fig. 6B show a severe effect of the cyt-5 mutations on cytochrome oxidase subunit III and cytochrome b mRNA levels. Amounts of coIII and cob mature mRNAs are severely reduced, as are the levels of most precursors. The observation that low levels of apparently correctly processed mRNA are present helps explain how the cyt-5 mutant strains can survive. There is a small increase in the levels of a 4.8-kb cob precursor RNA (Fig. 6B, 2), suggesting that an RNA processing enzyme or factor is deficient in the cyt-5 mutants.

The results of Fig. 6B, 1, are particularly surprising, as the coIII mRNA is probably transcribed from the same promoter as 19 S rRNA(24, 37) . The limiting factor(s) in mitochondrial rRNA accumulation apparently differ from those in mRNA accumulation. The differences between rRNA levels (almost unchanged) and mRNA levels (dramatically decreased) in cyt-5 mitochondria may reflect a difference in RNA stability. That is, rRNA levels appear to be nearly normal simply because rRNA is much more stable than mRNA. The data on mRNA levels in cyt-5 mutants suggest that mtDNA transcription is markedly decreased in these mutants.

RNA Polymerase Activity in the cyt-5 Mutants

To confirm that the cyt-5 mutations affect mtRNA polymerase, RNA polymerase activity was compared in crude mitochondrial lysates from wild-type and cyt-5 mutants. As shown in Fig. 7, lysates from wild-type (74A) and cyt-5 mutants containing the same amount of protein were assayed with a 19 S rRNA promoter-containing template. cyt-5-1 and cyt-5-4 mitochondrial lysates generated much lower levels of the expected 19 S transcript than did the wild-type lysate, suggesting a deficiency in transcription from this promoter. Note that the lanes containing mitochondrial lysates from cyt-5 mutants (Fig. 7, lanes 2 and 3) were exposed 7 times longer than the corresponding wild-type (lane 1). However, the size of the transcript was as expected (10) and no aberrant transcripts were visible in the products of this in vitro system, suggesting that correct initiation occurs in the cyt-5 mitochondrial lysates, but at low levels. Mixing experiments (Fig. 7, lane 6) suggest that the reduction in transcription activity is not due to the presence of an inhibitory component in cyt-5 lysates. The cyt-5 lysate may even stimulate transcription by the wild-type lysate (compare lanes 5 and 6 of Fig. 7). A possible explanation for this observation is that a component which is limiting in wild-type extracts (e.g. a transcription factor) is abundant in extracts prepared from cyt-5 mutants.

Figure 7: Transcription from the mitochondrial 19 S rRNA promoter is defective in cyt-5 mutants. Run-off transcription from the template pSRBP-1 digested with EcoRV gives an expected 325-nucleotide product. 1 µg of mitochondrial lysates from wild-type (lanes 1 and 5), cyt-5-1 mutant (lane 2), cyt-5-4 mutant (lane 3), or 1 µg each of wild-type and cyt-5-4 lysates (lane 6) were used in run-off transcription assays after pretreatment with antiserum against the major N. crassa endo/exonuclease. Lane 4 shows the activity of partially purified mtRNA polymerase from the poky strain of N. crassa in the absence of antiserum. Autoradiography was for 14 h (lanes 1 and 4) or 4 days (lanes 2 and 3). Size markers (not shown) were 5` end-labeled Sau3AI fragments of pBS(+).


As reported by others, we found it essential to include antiserum against the major N. crassa nuclease (10) in transcription reactions with crude mitochondrial lysates, otherwise transcripts or templates were completely degraded (not shown). We were able to partially purify mtRNA polymerase by heparin-Sepharose chromatography (10) from the poky mutant, where it is over-expressed (Fig. 7, lane 4). The activity of the purified mtRNA polymerase is easily detected in the absence of antiserum and yields a run-off transcript of the same size as do the crude extracts in the presence of antiserum, suggesting that the antiserum has no direct effect on mtRNA transcription. RNA polymerase activity in wild-type and cyt-5 mutant mitochondrial extracts was too low to permit purification of the mtRNA polymerase from these extracts.

DISCUSSION

The gene complementing cyt-5 mutants of N. crassa has been cloned and sequenced. The corresponding gene product is homologous throughout most of its length with S. cerevisiae mitochondrial RNA polymerase and, to a lesser degree, with genes encoding bacteriophage RNA polymerases. The N. crassa mtRNA polymerase retains the amino acids which are thought to be essential for the activity of all monomeric RNA polymerases. The invariant amino acids corresponding to Asp-900, Lys-969, Tyr-977, Gly-978, and Asp-1179 of the cyt-5 sequence (Fig. 3), are located in the template-binding cleft and form a putative catalytic pocket in the ``palm'' of the T7 RNA polymerase x-ray crystal structure(38) . These residues are part of three motifs which are highly conserved in a variety of different DNA and RNA polymerases(39) . Most of the strongly conserved domains found in mitochondrial and bacteriophage RNA polymerases (Fig. 3) line the template-binding cleft of T7 RNA polymerase.

There is very little sequence homology between the N-terminal 350 or so amino acids of yeast and N. crassa mtRNA polymerases (Fig. 3). The N terminus of T7 RNA polymerase is located quite far away from the template-binding region, and this polymerase remains functional even when a eukaryotic nuclear localization signal is attached at its N terminus(40) . Structural comparisons (41) between T7 RNA polymerase and the Klenow fragment of DNA polymerase I suggest that the N terminus of bacteriophage RNA polymerase (residues 1-307) folds as a subdomain to one side of the conserved palm, finger, and thumb subdomains(42) . The N-terminal highly variable extensions noted in the fungal mitochondrial RNA polymerases could simply enlarge this bulge and be accommodated in the T7 RNA polymerase three-dimensional structure without disrupting the active site. These variable N termini located some distance from the active site are candidates for transcription factor interaction sites. The observed sequence variability between the N termini of mtRNA polymerases presumably parallels changes that have occurred in the initiation specificity factor (43) and the mitochondrial promoter sequence(10, 44) .

The most striking difference between the cyt-5 gene product and other RNA polymerases in the same family is the presence of a stretch of polyglutamine. Polyglutamine segments are found in an enormous variety of proteins, and are one class of the sequence repeats called opa repeat elements(45) . TATA-binding proteins of different eukaryotic species have a highly conserved C-terminal domain and a divergent N-terminal domain which is required for transcriptional activation. Much of the variability in the TATA-binding proteins N-terminal domain is attributable to simple sequence repeats, some encoding stretches of polyglutamine(46, 47) . It has been suggested that the length variability within these repeated sequences is due to slippage by DNA polymerase, which has occurred independently in different lineages(47) . Once the slippage has occurred, putative advantageous properties of polyglutamine, including its possible involvement in protein-protein interactions(48) , lead to evolutionary selection for and retention of these repeats. In other genes, the polyglutamine repeat appears to be nonessential for function. For example, the nit-4 regulatory protein of N. crassa retains functional activity, based on its ability to transform a nit-4 mutant, after its polyglutamine sequence is deleted(49) . The stretch of polyglutamine in the N. crassa gene may function simply as a linker between the N and C termini, or it could be directly involved in specific protein-protein interactions. The region including the polyglutamine stretch in N. crassa mtRNA polymerase is required for complementation of the cyt-5 mutants, since removal of this sequence and an additional 114 amino acids by cleavage at the downstream BglII site abolishes complementing activity (Fig. 2).

The T7 RNA polymerase C terminus is adjacent to the catalytic pocket (38) , and is required for catalysis(50) . Residue Phe-882 of T7 RNA polymerase, corresponding to Phe-1421 in the cyt-5 sequence, is proposed to interact with the incoming rNTP. These findings add credence to the short stretch of conserved sequence we note at the C termini of bacteriophage and mitochondrial RNA polymerases (Fig. 3). Although the intact C terminus of the cyt-5 gene is required for high efficiency transformation, (^4)neither the C terminus nor the acidic domain near the C terminus are absolutely required for cyt-5 complementing activity (assuming transformation occurs via nonhomologous integration) (Fig. 2). It should be noted that both of the fungal mtRNA polymerases have a similar acidic insertion relative to bacteriophage RNA polymerases, but the one in N. crassa is considerably longer than that of yeast. Numerous nuclear transcription factors have acidic domains that are required for activity(48) . The acidic insertion in the mtRNA polymerases may likewise be involved in protein-protein interactions. This acidic insertion presumably replaces a long surface loop in the ``thumb'' domain of T7 RNA polymerase(38) .

In yeast, a single mtRNA polymerase is responsible for transcription of all coding sequences and very likely for priming of DNA replication (51) . It is likely that the mtRNA polymerase in Neurospora may function analogously. Although rRNAs accumulate essentially to the same levels in cyt-5 mutants as in the wild-type, we have shown that cob and coIII mRNA levels (Fig. 6), and transcription from the 19 S rRNA promotor (Fig. 7) are dramatically reduced in cyt-5 mutant lysates. These findings, together with the sequence homology noted above, support the identification of the cyt-5 gene product as the mitochondrial RNA polymerase responsible for rRNA and mRNA transcription.

The relative overabundance of a 4.8-kb cob pre-mRNA in the cyt-5 mutants (Fig. 6B, 2) is consistent with findings by others of a link between transcription and splicing in S. cerevisiae mitochondria. Dobinson et al. (24) suggested that the 4.8-kb pre-mRNA contains both introns found in the N. crassa cytochrome b gene. Its overabundance in cyt-5 mutants could be the manifestation of a deficiency in some other mitochondrial component (e.g. a mitochondrially synthesized component of the splicing apparatus) or it could reflect defective recruitment of such a component by the mtRNA polymerase transcription complex. The NAM1 gene product of S. cerevisiae is thought to interact with the mtRNA polymerase and has pleiotropic effects on transcription, splicing, and translation(52, 53) . If a factor with function similar to NAM1 exists in N. crassa, then a deficiency in mtRNA polymerase could lead directly to aberrant splicing of pre-mRNAs such as the 4.8-kb cob precursor.

Finally, immediately upstream of the cyt-5 gene lies another transcriptional unit of unknown function. The observation that transcription of this gene is induced in the poky mutant of N. crassa suggests that it also encodes a mitochondrial polypeptide (Fig. 4). We have mapped a gene fragment encoding part of a putative mitochondrial RNA helicase to linkage group IV, near the cyt-5 gene. (^5)Another cytochrome-deficient mutant of N. crassa, cyt-19, is also tightly linked to cyt-5(54) . Therefore, the cyt-5 gene may be located within a cluster of nuclear genes encoding mitochondrial products.

FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM39498 (to C. A. B.) and GM37949 (to A. M. Lambowitz). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L25087[GenBank].

§
Present address: Dept. of Biology, Hiram College, Hiram, OH 44234.

To whom correspondence should be addressed. Tel.: 614-292-9473; Fax: 614-292-6773.

(^1)
The abbreviations used are: kb, kilobase; cob, cytochrome b; coIII, cytochrome oxidase subunit III.

(^2)
J. Kennell, unpublished data.

(^3)
A. R. Kubelik, unpublished data.

(^4)
A. R. Kubelik, unpublished observation.

(^5)
B. Chen, unpublished data.

ACKNOWLEDGEMENTS

This work could not have been accomplished without constant encouragement and useful suggestions from Alan M. Lambowitz, to whom we are deeply indebted. We thank John Kennell for help with RNA polymerase assays. We acknowledge the generosity of Terry Chow (Montréal), who provided the antiserum against the N. crassa endo/exonuclease used in the RNA polymerase assays. We thank Georg Mohr for numerous helpful suggestions throughout the course of this work. The technical assistance of Amina Ahmed, Douglas R. Johnson, and Ying Tao is appreciated. We thank Alan Lambowitz and Roland Saldanha for critical comments on the manuscript. We acknowledge the use of the BIOSCI electronic newsgroup network, supported in Europe by the Seqnet facility at Daresbury, United Kingdom, and in the United States by the National Science Foundation with contributions from the Department of Energy and the National Institutes of Health.

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