Dual Targeting of Phage-Type RNA Polymerase to both Mitochondria and Plastids Is Due to Alternative Translation Initiation in Single Transcripts

• Published in: Biochemical and biophysical research communications Vol. 289; no. 5; pp. 1106 - 1113
• Main Authors: Kobayashi, Yuki, Dokiya, Yuko, Sugita, Mamoru
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.12.2001
• Subjects: Alternative Splicing; Amino Acid Sequence; Bacteriophage T7 - enzymology; Bacteriophage T7 - genetics; bacteriophage T7-type RNA polymerase; Base Sequence; cellular localization; DNA, Complementary - genetics; DNA, Plant - genetics; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; dual targeting; Exons; Genes, Plant; Introns; mitochondria; Mitochondria - enzymology; Molecular Sequence Data; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana sylvestris; NsRpoT-B gene; NsRpoTpm protein; Peptide Chain Initiation, Translational; Phylogeny; Plant Proteins; plastids; Plastids - enzymology; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Plant - genetics; RNA, Plant - metabolism; Sequence Homology, Amino Acid; Viral Proteins; bacteriophage T7-type RNA polymerase; Nicotiana sylvestris; dual targeting; cellular localization; mitochondria; plastids
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2001.6130

We isolated and sequenced a nuclear gene and cDNA encoding a bacteriophage T7-type RNA polymerase, NsRpoT-B, from Nicotiana sylvestris. The gene, NsRpoT-B, consists of 19 exons and 18 introns and encodes a polypeptide of 1020 amino acid residues. The predicted NsRpoT-B protein shows 71% amino acid identity with NsRpoT-A which is a mitochondrial protein. Quantitative RT-PCR revealed that steady-state NsRpoT-B mRNA accumulation is highest in the mature leaves and lowest in the cotyledons. Transient expression assays in protoplasts from N. sylvestris leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-B encodes dual targeting signals directing the protein into mitochondria and plastids. This strongly suggests that NsRpoT-B functions as an RNA polymerase transcribing genes from two different plant organelle genomes. NsRpoT-B transcripts have two potential translation initiation codons. An in vitro translation assay indicated that a chimeric mRNA encoding the N-terminal NsRpoT-B fused to an sGFP produced two polypeptides translated from the first and second initiation codons. This implies that the dual targeting of NsRpoT-B protein is regulated, in part, at the level of translation. We have designated this protein NsRpoTpm.