Virus-like particle formation and translational start site choice of the plant retrotransposon Tto1.
Ty1/copia group retrotransposon Tto1 from tobacco was put under control of an inducible promoter for expression in Arabidopsis thaliana. The system was used to analyze intermediates of the transposition process. The Tto1 RNA 5' region has a complex structure and contains several AUG codons. We therefore sought to experimentally define the translation initiation site. Constructs starting at various positions within the structural gag region were expressed in planta and functionally characterized. We found that gag proteins starting at the first ATG of the gag-pol ORF (ATG1), but also those starting at the third ATG of the gag-pol ORF (ATG3), can form virus-like particles (VLPs). However, gag protein expressed by the inducible Tto1 element had a size similar to gag starting at ATG1, and mutation of ATG1 in the inducible element abolished reverse transcription. This suggested that translation initiation at ATG1 is essential for the Tto1 life cycle. To support this conjecture, gag protein starting at ATG1, or gag protein shortened amino-terminally by nine amino acids (starting at the second ATG of the gag region, ATG2), was co-expressed with Tto1 carrying mutations at ATG1 and ATG2. Trans-complementation of the defective Tto element by gag starting at ATG1, but not by gag starting at ATG2, defines ATG1 as the functional translation initiation site.
Böhmdorfer
Gudrun
G
Max Planck Institute for Plant Breeding Research, Department of Plant Developmental Biology, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany. gudrun.boehmdorfer@gmi.oeaw.ac.at
Luxa
Kerstin
K
Frosch
Andrea
A
Garber
Karin
K
Tramontano
Andrea
A
Jelenic
Srecko
S
Weber
Martina
M
Bachmair
Andreas
A
D017360:Arabidopsis / Q000235:genetics / Q000821:virology; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015750:Genes, gag; D015751:Genes, pol; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D010942:Plant Viruses / Q000235:genetics* / Q000648:ultrastructure*; D010944:Plants / Q000235:genetics / Q000821:virology*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D018626:Retroelements / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics / Q000821:virology; D014771:Virion / Q000235:genetics* / Q000648:ultrastructure
D017931:DNA Primers; D018744:DNA, Plant; D004279:DNA, Viral; D018626:Retroelements