Synthetic and biochemical studies of retrotransposon Ty1.

摘要

The characterization of Ty1 retrotransposition and its component RNA genome and proteins will continue to extend our knowledge of the general mechanisms of retroelement replication that will likely be useful in future antiretroviral strategies. Synthetic and Biochemical studies of the Ty1 retrotransposon are described here. We show here the screening and characterization of suppressor mutants capable of increased transposition under conditions of elevated Mn +2 ion concentrations. Elevated Mn2+ inhibit in vitro Ty1 reverse transcriptase (RT) polymerizing activity, however, suppressors localize not to the polymerizing domain of RT itself but to the RNase H domain of the protein. In addition, Mn2+ inhibition of in vitro RT activity is greatly reduced in all the isolated suppressor mutants, whereas RNase H activity and cleavage specificity remain largely unchanged. We propose here that the effect of these suppressor mutations is transmitted to the polymerase domain and that biochemical communication occurs between these two domains during reverse transcription. Furthermore, we show that the in vivo inhibitory effects of elevated Mn 2+ are not limited to Ty1.We show that the in vivo activity of a hybrid Ty1/HIV-1 element, HART, can be reduced nearly 10-fold in the presence of elevated intracellular Mn2+. This result emphasizes the utility of Ty1 as a model organism in the study of retroviruses and is very suggestive that in vivo Mn2+ concentrations can be used to help regulate HIV-1 activity.;We design and construct two synthetic Ty1 elements here for the purpose of mapping novel Ty1 cis-acting sequences required for Ty1 transposition. One critical Ty1 sequence determinant is characterized and finely mapped here to a ∼70-bp region in the Ty1 reverse transcriptase gene. We show that synthetic sequence corresponding to the region results in a defect in Ty1 transposition as well as decreased Ty1 protein and RNA levels and that these defects can be rescued by restoring this region to native sequence. These results emphasize the utility of gene synthesis as a tool for extensive mutagenesis and Ty1 as a model for identification of novel cis-acting sequences for retroelement replication.