Our research program focuses on the mechanisms of retroelement action. To understand the complex functions required for the propagation of retroelements we study retrotransposons, a family of elements that are closely related to retroviruses. A significant advantage of studying retrotransposons is that 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 obtained strong evidence that Tf1 reverse transcriptase uses a novel mechanism of self-priming to initiate cDNA synthesis. This is in contrast to the tRNA mechanisms thought to be used by all other LTR-containing elements. The first 11 bases of the Tf1 transcript anneal to the primer binding site in a process that results in the initiation of reverse transcription. Primer extension assays and splinted ligation reactions were used to identify substantial amounts of the Tf1 transcript that were cleaved between the 11th and 12th bases. These results support the model that the first 11 bases of the Tf1 mRNA must be cleaved from the transcript to provide a 3'OH to serve as a primer for DNA synthesis. Mutations in the RNase H of Tf1 caused a sharp reduction in the accumulation of the cleaved product indicating that Tf1 RNase H may possess an unusual ability to recognize and cleave an RNA duplex. The overall importance of the self-priming mechanism was underscored by recent sequence analyses that indicated 7 other retroelements isolated from a variety of filamentous fungi probably utilize this self-priming mechanism. In each case strong complementarity exists between the primer binding site and the 5' region of the mRNA. The examination of the 5' untranslated region of the Tf1 mRNA revealed that this sequence possesses the potential to form a complex RNA structure composed of 39 basepairs that includes the primer binding site. This region of Tf1 was subjected to extensive mutagenesis that revealed that much of the structure contributes to transposition. The majority of the mutations in the structure resulted in a substantial reduction in the levels of cDNA produced. Second-site mutations that restored the complementarity of the structure resulted in elevated transposition frequencies and cDNA levels. These compensating mutations demonstrated that formation of the RNA structure is required for transposition and reverse transcription. Further analysis of the Tf1 RNA produced by elements with mutations in the RNA structure revealed that formation of the structure was required for the cleavage reaction that generated the primer. To characterize the Tf1 encoded factors required specifically for integration, we have screened a large set of mutant elements to identify those that are defective for transposition but nevertheless produce high levels of cDNA. Thus far, we have identified 7 mutations that reduce integration. Interestingly, two were found to be in RNase H, an enzyme thought to be required only for reverse transcription. Quantitative analysis indicated these mutations appear to have no effect on the levels of cDNA that accumulate. These results suggest that RNase H may provide a function that is directly required for integration.
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