Retroviruses and long terminal repeat-containing retroelements use host-encoded tRNAs as primers for the synthesis of minus strong-stop DNA, the first intermediate in reverse transcription of the retroelement RNA. Usually, one or more specific tRNAs, including the primer, are selected and packaged within the virion. The reverse transcriptase (RT) interacts with the primer tRNA and initiates DNA synthesis. The structural and sequence features of primer tRNAs important for these specific interactions are poorly understood. We have developed a genetic assay in which mutants of tRNA i Met , the primer for the Ty1 retrotransposon of Saccharomyces cerevisiae, can be tested for the ability to serve as primers in the reverse transcription process. This system allows any tRNA mutant to be tested, regardless of its ability to function in the initiation of protein synthesis. We find that mutations in the T⌿C loop and the acceptor stem regions of the tRNA i Met affect transposition most severely. Conversely, mutations in the anticodon region have only minimal effects on transposition. Further study of the acceptor stem and other mutants demonstrates that complementarity to the element primer binding site is a necessary but not sufficient requirement for effective tRNA priming. Finally, we have used interspecies hybrid initiator tRNA molecules to implicate nucleotides in the D arm as additional recognition determinants. Ty3 and Ty1, two very distantly related retrotransposons, require similar molecular determinants in this primer tRNA for transposition.The Ty1 element of the yeast Saccharomyces cerevisiae is a 6-kb retrotransposon with long terminal repeats (LTRs) at each end (10). Transcription of the element produces an RNA molecule from which the element-encoded proteins TYA and TYB are translated. These proteins are functional homologs of retroviral Gag and Pol proteins, respectively. The TYA and TYB proteins assemble into structures termed virus-like particles (VLPs) because of their similarity in structure to retrovirus core particles. These VLPs are intermediates in transposition within which reverse transcription occurs (17). During the assembly process, the Ty1 RNA is packaged within the VLPs and subsequently reverse transcribed into a full-length cDNA. In the final step of transposition, the cDNA is integrated into a new site in the host genome, and the cycle begins again with transcription of the element.Retroviruses and LTR retrotransposons share the problem of having to generate a full-length cDNA from a message that initiates and ends within the LTRs of the element (diagrammed in Fig.
The yeast Ty1 LTR retrotransposon replicates by reverse transcription and integration; the process shows many similarities to the retroviral life cycle. However, we show that plus strand strong-stop DNA transfer in yeast Ty1 elements differs from the analogous retroviral process. By analysis of the native structure of the Ty1 primer binding site and by a series of manipulations of this region and assessment of the effects on retrotransposition, we show that primer binding site inheritance is not from the tRNA primer, which is inconsistent with classical retroviral models. This unusual inheritance pattern holds even when the Ty1 primer binding site is lengthened in order to be more retrovirus-like. Finally, the distantly related Ty3 element has an inheritance pattern like Ty1, indicating evolutionary conservation of the alternative pathway used by Ty1. Based on these results we arrive at a plus strand primer recycling model that explains Ty1 plus strand strong-stop DNA transfer and inheritance patterns in the primer binding site.
Yeast retrotransposon Tyl transposes through an RNA intermediate by a mechanism resembling retroviral replication. Long terminal repeat retroelements require primers for initiation of reverse transcription. The primer for minus-strand DNA synthesis is the 3' end of a cellular tRNA that base pairs to the complementary region of genomic RNA (the primer binding site). The genomic RNA of retroviruses and retrotransposons is shorter than its proviral DNA counterpart, lacking complete long terminal repeats. A variety of models have been proposed to describe how complete long terminal repeats are regenerated during reverse transcription. A common feature of these models is the requirement that the 3' portion of the primer tRNA be reverse-transcribed and then utilized in a strand-transfer reaction. We introduced a silent mutation into the Tyl primer binding site and followed its fate during a single cyde of reverse transcription to directly test this aspect of the reverse transcription model. We demonstrate that the tRNA sequence is not inherited by progeny Tyl elements during reverse transcription.Tyl, a long terminal repeat (LTR)-containing retrotransposon in the yeast Saccharomyces cerevisiae, transposes through an RNA intermediate (1). In this way, the Tyl life cycle resembles that of retroviruses. Like most DNA polymerases, Tyl-encoded reverse transcriptase (RT) is primerdependent. In LTR retroelements, the synthesis of minusstrand DNA is primed by an element-specific cellular tRNA (2). The Tyl primer is the initiator tRNAMet (3).The RNA genomes of LTR retroelements contain a region complementary to the 3' end of primer tRNA called the primer binding site (PBS), located just downstream of the 5' LTR (2). The precise position of priming by tRNA is very important because it determines the 3' DNA end of the new DNA copy (Fig. 1) (2, 5). Genomic RNAs of retroviruses and LTR retrotransposons are shorter than proviral DNA, having just small terminal repeats at each end (see Fig. 1 for LTR domain nomenclature). Full-length DNA products with complete LTRs are regenerated during reverse transcription, a conserved process in LTR retroelements (2).A variety of models describe how the ends of LTR retroelements are regenerated during DNA synthesis (6, 7). According to these models, the minus-strand strong-stop DNA, the first discrete intermediate in DNA synthesis, is completed when tRNA-primed synthesis reaches the 5' end of the template RNA. The RNA portion of the resulting RNA-DNA hybrid is digested by the RT-associated RNase H. Nascent minus-strand strong-stop DNA, consisting of R-U5 sequences attached to primer tRNA (Fig. 1), is transferred and annealed to the 3' end of the genomic RNA via its complementary small terminal repeat sequence and can then be extended to the 5' end of this template. Plus-strand DNA synthesis commences from an RNase H-resistant oligoribonucleotide region at the upstream boundary ofthe 3' LTR, in PPT1. RT then proceeds toward the tRNA molecule covalentlyjoined to the minus-strand DNA templa...
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