Dual targeting of phage-type RNA polymerase to both mitochondria and plastids is due to alternative translation initiation in single transcripts

Biochem Biophys Res Commun. 2001 Dec 21;289(5):1106-13. doi: 10.1006/bbrc.2001.6130.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing
  • Amino Acid Sequence
  • Bacteriophage T7 / enzymology
  • Bacteriophage T7 / genetics
  • Base Sequence
  • DNA, Complementary / genetics
  • DNA, Plant / genetics
  • DNA-Directed RNA Polymerases / genetics*
  • DNA-Directed RNA Polymerases / metabolism*
  • Exons
  • Genes, Plant
  • Introns
  • Mitochondria / enzymology*
  • Molecular Sequence Data
  • Peptide Chain Initiation, Translational
  • Phylogeny
  • Plant Proteins*
  • Plastids / enzymology*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Sequence Homology, Amino Acid
  • Tobacco / enzymology*
  • Tobacco / genetics*
  • Viral Proteins

Substances

  • DNA, Complementary
  • DNA, Plant
  • Plant Proteins
  • RNA, Messenger
  • RNA, Plant
  • Viral Proteins
  • NsRpoT-B protein, Nicotiana sylvestris
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases

Associated data

  • GENBANK/AB058957
  • GENBANK/AB058958