Dual targeting of phage-type RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts.
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.
Kobayashi
Y
Y
Center for Gene Research, Nagoya University, Nagoya, 464-8602, Japan.
Dokiya
Y
Y
Sugita
M
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D005091:Exons; D017343:Genes, Plant; D007438:Introns; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010802:Phylogeny; D010940:Plant Proteins; D018087:Plastids / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*; D014764:Viral Proteins
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D014764:Viral Proteins; C446921:NsRpoT-B protein, Nicotiana sylvestris; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases