Our research program focuses on the mechanisms of retroelement action. Our approach to understanding the complex interactions between the retroelement and its host is to study retrotransposons, a family of elements that are closely related to retroviruses. A significant advantage to studying retrotransposons is they exist in hosts such as yeast that can readily be studied using sophisticated molecular genetic techniques. In the process of characterizing yeast transposition, we have collected strong evidence that Tf1 reverse transcriptase uses a novel self-priming mechanism to initiate cDNA synthesis. This is in complete contrast to the tRNA mechanisms thought to be used by all other LTR- containing elements. In this report we describe the characterization of the minus-strand strong-stop DNA that provides additional support for the self-priming mechanism. Genetic and biochemical analysis of Tf1 RT mutations in the active site of the polymerase allowed us to observe priming intermediates consisting of transcripts that had the first 11 bases removed. This data suggested a molecular model for priming that includes a cleavage of the first 11 bases of the transcript and the priming of reverse transcription from the 3'OH of the 11th base. The analysis of a large family of point mutations near the primer binding site (PBS) confirmed the presence of a new 39 base pair RNA structure that is essential for transposition. As this large structure includes the 11 base pairs shown to be important for priming, we speculate that the newly detected structure may also participate in the self-priming mechanism. The assembly of functional Tf1 particles has been a paradox since other retroelement particles assemble with a molar excess of capsid protein that accumulates because the levels of the Pol proteins are restricted by reading frameshifts or stop codons. Tf1 however, expresses all its protein from within a single open reading frame as a primary translation product and we found that Tf1 particles contain a 26-fold excess of Gag compared to IN protein. By looking at cultures in different stages of growth, we have been able to observe an IN degradation process that leads to this excess of Gag. In addition, we found that most of the IN degradation occurred before Tf1 cDNA is synthesized indicating that the particles with a 26-fold excess of Gag are intermediates in transposition. We have used immunoblot analysis to reveal that IN degradation occured in cells starved for glucose and not in those cell starved for nitrogen, suggesting that the loss of IN is not a time-dependent process but occurred only after certain growth conditions are met. To determine the factors required for transposition, we have created a large set of mutant strains that are defective for transposition. Thus far, we have identified six host genes that are required for either protein accumulation, particle stability, or integration.
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