A Ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization

Reverse transcriptases (RTs) are found in a Ride variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar int rons. An invariant triad of aspartates is thought to be required for the ca talytic function of RTs. We generated RT mutants in the yeast retrotranspos on Ty1, changing each of these active-site aspartates to asparagine or glut amate. All but one of the mutants lacked detectable polymerase activity. Th e novel exception, D211N, retained near wild-type in vitro polymerase activ ity within virus-like particles but failed to carry out in vivo transpositi on. For this mutant, minus-strand synthesis is impaired and formation of th e plus-strand strong stop intermediate is eliminated. Intragenic second-sit e suppressor mutations of the transposition defect map to the RNase H domai n of the enzyme. Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical. This impl ies a basic difference in the polymerase active-site geometry of Ty1 and hu man immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzy me.