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To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5 end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.Mutations in the PRT1 gene of the yeast Saccharomyces cerevisiae, as exemplified by the temperature-sensitive prt1-1 mutant allele, cause a conditional impairment of translation initiation in vivo (26, 27; see also reference 18); in vitro, prt1-1 mutant cell extracts are defective for the interaction of the eIF-2 GTP Met-tRNA i ternary complex with the 40S ribosomal subunit (16), suggesting that the PRT1 polypeptide (Prt1p) may be a component of the yeast translation initiation factor 3 (eIF-3) complex (37, 38). Recently, this suggestion has been validated by the identification of Prt1p in purified yeast .Mutant forms of the PRT1 gene have been found several times by selection for temperature-sensitive growth mutants (26,27,58) and also through searches for more-specific effects. The prt1-63 allele (formerly cdc63 [22]) was obtained by selection for mutants conditionally unable to perform the G 1 cell cycle regulatory step (2), whereas the prt1-26 allele (formerly dna26 [15]) was identified in a mutant defective for DNA synthesis (13). The identification of prt1 mutant alleles by the latter criteria can be understood from the effects of prt1 mutations on the cell cycle. At appropriate restrictive temperatures, all temperature-sensitive prt1 mutations can cause regulated cell cycle blockage (23), and the DNA synthesis impairment caused by the prt1-26 mutation was shown to be an indirect consequence of a concerted arrest of mutant cells in the G 1 phase of the cell cycle (15). Thus, prt1 mutant alleles can allow sufficient protein synthesis for completion of an ongoing cell cycle (7) while blocking initiation of the next cell cycle and DNA replication.Prt1p has been characterized by analysis of the cloned PRT1 gene (24). In this report, we extend the characterization of Prt1p by showing that the open reading frame (ORF) of PRT1 does not accurately predict Prt1p and by identi...

We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.