Accurate transcription of a plant mitochondrial gene in vitro.

Abstract

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.

Arabidopsis Phage-Type RNA Polymerases: Accurate in Vitro Transcription of Organellar Genes

“…The high molecular weight signals that are visible at the top of the autoradiograph may in part be attributed to transcripts initiated nonspecifically on the plasmid template at sequences other than the atp6-1 promoters; another part likely represent RNAs not terminated at hisa and thra. Nonspecific high molecular weight transcripts were likewise seen in in vitro transcription studies with mitochondrial extracts (Hanic-Joyce and Gray, 1991;Rapp and Stern, 1992;Binder et al, 1995). The minor signals indicated by gray arrows and marked with asterisks were assumed to correspond to differently migrating major transcripts rather than additional defined RNA 59 ends, as no discrete fragments other than those resulting from initiation at Patp6-1-200 were detected by 59-rapid amplification of cDNA ends (RACE) (Figure 3).…”

A tRNA gene transcription initiation site is similar to mRNA and rRNA promoters in plant mitochondria

“…Only a specific endonuclease can release this spacer molecule with a precise 5' terminus during processing (27).…”

A Novel Pea Mitochondrial in Vitro Transcription System Recognizes Homologous and Heterologous mRNA and tRNA Promoters

“…The isolation of such transcriptionally active extracts from dicot plant mitochondria was additionally guided by the successful development of an in vitro transcription system for monocot plants (Hanic-Joyce and Gray, 1991;Rapp and Stern, 1992). Because data on functional transcription initiation sites were only available for Oenothera and soybean, the isolation of transcriptionally active protein fractions was started in these two plant species.…”

“…The successful development of in vitro transcription systems for the monocot plant species wheat and maize confirms the competence of several promoters of protein coding genes in vitro (Hanic-Joyce and Gray, 1991;Rapp and Stern, 1992). Detailed investigation by deletion analysis, linker-scanning mutagenesis, and site-directed mutagenesis revealed the atp1 promoter in maize to be composed of a central domain extending from Ϫ7 to ϩ5 and a 3-base pair upstream domain located between positions Ϫ10 and Ϫ12.…”