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from Contents Ty1 background | reverse transcription | yeast assay S. cerevisiae contains five families of long terminal repeat (LTR) retrotransposons, Ty1-Ty5. Transcription of Ty1 produces an RNA molecule from which the element-encoded proteins TYA and TYB are translated (Figure 1).
Figure 1: The basic structure of Ty1 DNA is diagrammed, with the LTRs indicated by the filled-in triangles. The TYA and TYB proteins are indicated as straight lines below the element, and the approximate location of the PR, IN and RT coding regions within TYB are indicated. These proteins are functional homologs of retroviral gag and pol, respectively. TYA encodes a capsid-like protein, and TYB encodes enzymes required for specific steps in the retroviral life-cycle: protease (PR), integrase (IN), and reverse transcriptase/RNAseH (RT). The stop codon at the end of the TYA ORF is occasionally bypassed by a frameshifting event at the TYA/TYB junction, resulting in a TYA-TYB fusion protein. During the Ty1 "life-cycle", the TYA and TYA-TYB proteins assemble into structures termed virus-like particles (VLPs), due to their similarity in structure to retrovirus core particles (figure 2). These VLPs are intermediates in transposition within which the TYA and TYB proteins are processed by protease and reverse transcription occurs. During the assembly process, the Ty1 RNA is packaged within the VLPs and subsequently reverse transcribed by RT into a full-length cDNA. In the final step of transposition, integrase protein mediates integration of the cDNA into a distant site in the host genome, and the cycle begins again with transcription of the element. Figure 2: The Ty1 life-cycle (back to text) Reverse Transcription Retroviruses and LTR-retrotransposons share the problem of having to generate a full-length cDNA from a message that initiates within the LTRs of the element. This is accomplished by initiating reverse transcription near the 5’ end of the element, immediately next to the LTR, in a region termed the primer binding site (PBS) (Figure 3). This produces a short DNA, termed minus strong-stop DNA, part of which is complementary to the 3’ end of the RNA within the repeat (R) region. The minus-strong stop DNA is transferred to the 3’ end of the RNA, where it serves as the primer for more extensive minus-strand synthesis.  Figure 3. Reverse transcriptase (RT) priming. The rectangle diagrams Ty elemental DNA with the black triangles indicating the orientation of the LTRs. The U3 (unique to 3’ end of RNA), R (repeated in the RNA) and U5 (unique to 5’ end of RNA) regions of the LTR are indicated. The long right-facing arrow represents RNA. The complementarity between the RNA primer binding site (PBS) and the primer tRNA is diagrammed. The short left-facing arrow represents minus strong-stop DNA. The reverse transcriptases (RTs) of retroviruses and LTR-retrotransposons use host-encoded tRNAs as primers for synthesis of minus strong-stop DNA. The particular tRNA used varies according to the particular retroelement. Ty1 and Ty3 use the tRNAiMet as a minus strong-stop primer. The last 10 nucleotides of the acceptor stem of tRNAiMet are complementary to the Ty1 PBS (Figure 3). The tRNAiMet is specifically packaged by an unknown mechanism into Ty1-VLPs. For Ty1 it has been shown that the priming tRNAiMet is 10- to 40-fold enriched in VLPs compared with the cytoplasmic levels of this tRNA. This complementarity allows tRNAiMet to prime synthesis of minus strong-stop DNA. Due to the genetical limitations of larger eukaryotic experimental systems, studies on the interaction of primer tRNA with RT in the context of maturing virions is very difficult. However, the genetics available in yeast allow this interact to be studied in vivo, in the context of maturing Ty virus-like particles. The lab is currently making mutations in the tRNA primer and using an in vivo transposition assay to genetically select mutations affecting transposition. Yeast system to test tRNA mutants for transposition In vivo studies of the effect of primer tRNA mutants on retrotransposition are complicated by the presence of the wild-type primer tRNA, encoded by multiple genes, in the genome of the host cell. Employing genetic techniques available for yeast, we have developed a system in which any mutant tRNAiMet can be tested in vivo for its ability to support Ty1 transposition. The assay system uses a strain in which all four genomic copies of IMT have been disrupted by TRP1. (Bystrom et al) The only functional IMT gene present in these cells is supplied on an extrachromosomal plasmid. A key to this system is that any imt mutation can be tested for its ability to support Ty1 transposition, regardless of its ability to function as an initiator tRNA. This was accomplished by constructing a mutation, imt4-9, in which the complementarity between the tRNA acceptor stem and the Ty1 PBS has been disrupted (figure 4). Although imt4-9 will not support transposition, it can initiate translation effectively; thus, a strain relying solely on imt4-9 grows readily (Chapman et al).
Figure 4. The nucleotide sequence of the tRNAiMet (IMT4) acceptor stem (3' end complementary to the primer binding site (PBS)) is shown. The residues mutated to generate imt4-9 are indicated. The strain genotype and plasmids used in the transposition assay are diagrammed
in Figure 5: Schematic drawing of the transposition assay
The presence of imt4-9 assures that any mutation can be tested, even if it is not translationally competent. Due to a red pigment produced by ade2 mutants, the translational function of any mutant is easily observed by plating to rich media and observing red sectoring, indicating loss of imt4-9. |
