The protease and reverse transcriptase of the tobacco LTR retrotransposon Tnt1 are enzymatically active when expressed in Escherichia coli

The open reading frame (ORF) of the tobacco retrotransposon Tnt1-94 was ove r-expressed in Escherichia coli to assay its protease and reverse transcrip tase (RT) enzymatic activities. In E. coli, Tnt1-94 polyprotein is cleaved off by the element-encoded protease to release a Gag protein with an appare nt molecular mass of 37 kDa that forms high-density aggregates. The catalyt ic site of Tnt1-94 protease (D-T-A) as determined by deletion analysis diff ers 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 a ctivity. 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.