title: Cauliflower mosaic virus reverse transcriptase. Activation by proteolytic processing and functional alteration by terminal deletion.
abstract: We have previously expressed the cauliflower mosaic virus (CaMV) reverse transcriptase (RTase) gene, the ORFV gene, in yeast in an active form (RTase-Y). An activity gel analysis revealed that the molecular size of RTase-Y as well as an RTase associated with the CaMV particles (RTase-V) is 60 kDa. This size is about 18 kDa smaller than that of the inactive form previously expressed in Escherichia coli (RTase-E) (78 kDa), which corresponds to the coding capacity estimated for the ORFV gene. To investigate the possible involvement of proteolytic processing in the de novo synthesis of CaMV RTase, we constructed a series of deletions from either terminus or both termini of the ORFV coding sequence and expressed them in E. coli. Among the various truncated RTases, those (denoted delta N) that lack N-terminal peptide fragments 143-185 amino acids long were active on the synthetic RNA template-primer, poly(rC)-oligo(dG). Those RTases (denoted delta C) lacking C-terminal peptide fragments 50-102 amino acids long and those lacking both termini (denoted delta NC) were also active on this template. However, only the delta N RTases showed enzyme properties indistinguishable from the RTase-Y in that they transcribed natural RNA into DNA and required either Mg2+ or Mn2+ for their activity. The length of the deletion corresponded approximately to the difference of the molecular weights between RTase-Y and RTase-E. These results suggest that CaMV RTase is translated in an inactive precursor form and then converted to an active form by proteolytic processing during de novo synthesis. We have also demonstrated that C-terminal deletions cause a loss of activity on a natural RNA template accompanied by an alteration in metal ion requirement. The inability to incorporate dTTP accounts for the loss of activity on the natural RNA template. However, the affinities for dTTP and the corresponding template, poly(rA)-oligo(dT), were found to be unaltered.
authors.affiliation: Laboratory of Molecular Genetics, National Institute of Agrobiological Resources, Ibaraki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Takatsuji
authors.forename: H
authors.initials: H
authors.lastname: Yamauchi
authors.forename: S
authors.initials: S
authors.lastname: Watanabe
authors.forename: H
authors.initials: H
authors.lastname: Kato
authors.forename: J E
authors.initials: JE
authors.lastname: Ikeda
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent; D002872:Chromosome Deletion; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006868:Hydrolysis; D009029:Mosaic Viruses / Q000201:enzymology*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D012194:RNA-Directed DNA Polymerase