Reverse transcription of the yeast Ty1 retrotransposon is primed by tRNAi(Met) base paired to the primer binding site near the 5'-end of Ty1 genomic RNA. To understand the molecular basis of the tRNAi(Met)-Ty1 RNA interaction the secondary structure of the binary complex was analysed. Enzymatic probes were used to test the conformation of tRNAi(Met) and of Ty1 RNA in the free form and in the complex. A secondary structure model of the tRNAi(Met) Ty1 RNA complex consistent with the probing data was constructed with the help of a computer program. The model shows that besides interactions between the primer binding site and the last 10 nt at the 3'-end of tRNAi(Met), three short regions of Ty1 RNA named boxes 0, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Mutations were made in the boxes or in the complementary sequences of tRNAi(Met) to study the contribution of these sequences to formation of the complex. We find that interaction with at least one of the two boxes 0 or 1 is absolutely required for efficient annealing of the two RNAs. Sequence comparison showing that the primary sequence of the boxes is strictly conserved in Ty1 and Ty2 elements and previously published in vivo results underline the functional importance of the primary sequence of the boxes and suggest that extended interactions between genomic Ty1 RNA and the primary tRNAi(Met) play a role in the reverse transcription pathway.

LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5' and 3' ends of Ty1 genomic RNA interact through 14 nucleotide 5'-3' complementary sequences (CYC sequences). This 5'-3' base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5'-3' interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.

Retroelements are mobile genetic entities that replicate via reverse transcription of a template RNA. A key component to the life cycle of these elements is the enzyme reverse transcriptase (RT), which copies the single-stranded genomic RNA of the element into a linear double-stranded DNA that is ultimately integrated into the host genome by the element-encoded integrase. RT is a multifunctionnal enzyme which possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity that specifically degrades the RNA strand of RNA-DNA duplexes. At some stages of the replication a strand-displacement activity of RT is also necessary. All activities are essential for the conversion of single-stranded genomic RNA into the double-stranded preintegrative DNA. This review focuses on the role of RT in the different steps of the replication process of retroelements. The features of retrotransposon replication which differ from the retroviral ones will be emphasized. In a second part of the review, the biochemical and enzymatic properties of two newly characterized retrotransposon RTs will be described. The role of the integrase domain in reverse transcriptase activity of some retroviral and retrotransposon RTs will be discussed.

Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.