(Received for publication, November 16, 1995)
From the
The Neurospora crassa mutants, cyt-5-1 and cyt-5-4, have a cytochrome b- and aa-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.
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, 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.
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 (
)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.
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) .
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.
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 -tubulin
probe. For the experimental gels, the amount of RNA in each lane was
adjusted to yield equivalent amounts of
-tubulin mRNA, as
determined by the intensity of the corresponding band on the trial gel.
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
-peaks of
cytochromes c, b, and aa
,
respectively.
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
+.
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
(
) 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.
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 -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
-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.
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
-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.
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.
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, ()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. ()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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L25087[GenBank].