The open reading frame (ORF) of the tobacco retrotransposon Tnt1-94 was over-expressed in Escherichia coli to assay its protease and reverse transcriptase (RT) enzymatic activities. In E. coli, Tnt1-94 polyprotein is cleaved off by the element-encoded protease to release a Gag protein with an apparent molecular mass of 37 kDa that forms high-density aggregates. The catalytic site of Tnt1-94 protease (D-T-A) as determined by deletion analysis differs from that of retroviruses and of well-characterized retrotransposons (D-T/S-G). The cleaved or uncleaved ORF of Tnt1-94 displays an exogenous RT activity. Over-expression of plant retrotransposons ORFs in E. coli provides a very useful strategy to assay the enzymatic activities of their proteins and to determine their catalytic sites.
Plant Molecular Biology – Springer Journals
Published: Oct 3, 2004
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