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PMID: 10567537 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria.

Molecular and cellular biology ·Vol. 19 ·No. 12 ·1999-12-00 ·Pages 8113-22

Ikeda TM, Gray MW

Abstract

To investigate the transcriptional apparatus in wheat mitochondria, mitochondrial extracts were subjected to column chromatography and protein fractions were analyzed by in vitro transcription and mobility shift assays. Fractions eluting from DEAE-Sephacel between 0.2 and 0.3 M KCl displayed DNA-binding activity and supported specific transcription initiated from a wheat cox2 promoter. The active DEAE-Sephacel pool was further resolved by chromatography on phosphocellulose. Fractions that exhibited DNA-binding activity and that stimulated both specific and nonspecific transcription in vitro were highly enriched in a 63-kDa protein (p63). From peptide sequence obtained from purified p63, a cDNA encoding the protein was assembled. The predicted amino acid sequence (612 amino acid residues, 69 kDa) contains a basic N-terminal targeting sequence expected to direct transport of the protein into mitochondria. The p63 sequence also features an acidic domain characteristic of transcriptional activation factors, as well as sequence blocks displaying limited similarity to positionally equivalent regions in sigma factors from eubacteria related to mitochondria. Recombinant p63 possesses DNA-binding activity, exhibiting an affinity for the core cox2 promoter element and upstream regions in gel shift assays and having the ability to enhance specific transcription in vitro. Transcripts encoding p63 are expressed at an early stage in the germination of isolated wheat embryos, in a temporal pattern parallelling that of newly synthesized precursors of cox2, a mitochondrial gene. Taken together, these data suggest a role for p63 in transcription in wheat mitochondria.

MeSH Terms
Amino Acid Sequence Chromosome Mapping Cloning, Molecular DNA, Mitochondrial/metabolism DNA, Plant DNA-Binding Proteins/genetics,isolation & purification,metabolism Gene Expression Genes, Plant Genome, Plant Germination Mitochondria/genetics,metabolism Molecular Sequence Data Plant Proteins/genetics,isolation & purification,metabolism Recombinant Fusion Proteins/genetics,isolation & purification,metabolism Sequence Homology, Amino Acid Transcription, Genetic Triticum/genetics
Chemicals
DNA, Mitochondrial DNA, Plant DNA-Binding Proteins Plant Proteins Recombinant Fusion Proteins
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Ikeda T M
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Gray M W
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.12.8113
Published
1999-12-00
Pages
8113-22
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC84896
Subset
IM
Databases
GENBANK
AF091837
External Links
PubMed source DOI: 10.1128/MCB.19.12.8113 PMC Full Text View journal details
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