We isolated a 63-kDa protein (p63) from wheat mitochondria on the basis of its ability to bind DNA and enhance transcription in an in vitro assay. In combination with 5′ RACE and heminested PCR, we assembled about 2.1 kbp of cDNA sequence encoding p63. Northern blot analysis revealed a transcript of about 2.4 kb (Fig. A), and so we expect that the 5′ untranslated region extends beyond the sequence that we obtained. Southern blot analysis suggests that the p63 gene is present in at least three copies in the wheat nuclear genome, whereas analysis of individual full-length PCR clones provided evidence of at least two major p63 sequence variants (data not shown). Considering the hexaploid nature of the wheat genome, the p63 coding sequence could be present as a single-copy gene in each of its constituent genomes (A, B, and D). The predicted amino acid sequence of p63 contains a typical N-terminal signal peptide required for transport of the protein into mitochondria; in fact, a computer-assisted analysis (47
) of this sequence clearly predicts that p63 should be targeted to mitochondria. Recombinant p63 expressed from the cDNA clone possesses DNA-binding activity, displaying weak affinity for the core promoter of the wheat cox2
gene and upstream regions (in maize, a close relative of wheat, a core promoter sequence and an upstream region both contribute to optimal transcriptional activity [9
]). These results suggest that recombinant p63 binds to a region of DNA directing specific transcription. However, although we conducted footprinting (DNase I protection) and exonuclease III protection studies to define a precise protein-binding DNA sequence in the vicinity of the cox2
promoter, we were unable to detect any specific region protected either by active DS fractions or by recombinant p63.
We did find a difference between native and recombinant p63 proteins in their effect on in vitro transcription: whereas fractions containing native p63 stimulated both nonspecific and specific transcription, recombinant p63 appeared to enhance specific transcription and suppress nonspecific transcription. Fractions containing partially purified, native p63 may well contain other factors involved in or influencing transcription in the in vitro assay. Because all of the transcriptionally active DS fractions that we tested supported specific as well as nonspecific transcription in vitro, it is not clear at this point whether p63 functions directly as a specificity factor that is essential for basal transcription or as an activator that enhances transcription but is not itself required for basal function. Preparation of a core RNA polymerase fraction devoid of transcription factors will be necessary before we can distinguish between these two possibilities.
Transcripts encoding p63 are present at a very low level in dry wheat embryos but accumulate to readily detectable levels by 5 h after the start of imbibition (Fig. A). Unprocessed cox2
transcripts (synthesized de novo) also appear in detectable amounts between 1 to 5 h following embryo imbibition (Fig. B); thus, the expression of nucleus-encoded p63 correlates temporally with de novo transcription of a mitochondrial gene during germination. This lends credence to the idea that p63 may be involved in the regulation of transcription of mitochondrial genes, as is mtTFA in vertebrate mitochondria (40
). Because we used isolated embryos lacking endosperm and seed coat for this analysis, the apparent decrease in transcript levels after 5 h of imbibition may not represent the actual situation in intact wheat seeds during germination. Further analysis will be necessary to clarify the relationship between the expression of p63 and wheat mitochondrial biogenesis.
In searches of public-domain protein databases, we could not detect any similarities between p63 and proteins known to be involved in transcription in mitochondria, including yeast mtTFB and human mtTFA. In view of evidence supporting the eubacterial origin of mitochondria (24
) and reports of a limited similarity of yeast mtTFB to eubacterial sigma factors (36
), we considered the possibility that p63, characterized here as a candidate transcription factor, might be distantly related to eubacterial sigma factors. Indeed, the local sequence similarities deduced between the wheat mitochondrial p63 sequence and eubacterial RpoD sequences (Fig. ), coupled with the positional correspondence of these regions within the compared proteins, support this possibility. On the other hand, we could not discern clear similarities in other regions conserved among different sigma factors, including the helix-turn-helix DNA-binding motif in regions 3 and 4.2 (33
Recently, Lang et al. (39
) reported that in the protist Reclinomonas americana
, the mitochondrial genome carries a set of genes (rpoA
) that encode the subunits that comprise a eubacterium-type RNA polymerase (α2
ββ′ς). Because the R. americana
mitochondrial genome appears to be an ancestral type of mtDNA, representative of an early stage in the evolution of this organellar genome (26
), one might expect that the mtDNA-encoded sigma factor would have been recruited as a transcription factor for mitochondrial gene expression, even after the evolutionary replacement of the mtDNA-encoded, eubacterium-like RNA polymerase by the nucleus-encoded, phage-type enzyme that now functions as the mitochondrial RNA polymerase in virtually all eukaryotes (11
). However, in our alignment, the p63 sequence does not show appreciably higher similarity to the R. americana
mitochondrial RpoD, which lacks the acidic domain and region 3.1, than to the eubacterial RpoD sequences (Fig. ). More detailed biochemical analysis will be required to explore the functional relevance of these similarities. Given the overall low level of similarity, it may be significant that a mutagenesis study of the yeast mtTFB and amino acid sequence comparison with a mtTFB homolog from closely related species both suggest that the mechanism of promoter recognition by the fungal mtTFB is different from that used by eubacterial sigma factors (10
Two promoter-specific DNA-binding proteins (34 and 44 kDa) have been identified in the mitochondria of pea, a dicotyledonous plant (31
). As noted above, our sequence similarity searches have identified Arabidopsis
protein sequences bearing significant similarity to the wheat p63 sequence. Whether either of the pea proteins is homologous to the wheat p63 and Arabidopsis
proteins is not known at this time. Because the core promoter sequence differs substantially between dicotyledonous and monocotyledonous plants, it would not be surprising if the corresponding RNA polymerases required different transcription factors, although similarities in the basal transcription machinery might be expected. In maize, a nonconsensus promoter has been found in lines having Zea perennis
); whether this alternative promoter interacts with a different transcription factor(s) than does the standard promoter remains to be ascertained. Further analysis will be necessary to definitively identify the minimal protein components essential for specific transcription in plant mitochondria and to better define differences in this process in monocotyledonous and dicotyledonous plants.