| 1. |
- Åström, S U, et al.
(författare)
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Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae
- 1999
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Ingår i: Molecular General Genetics. - : Springer. - 0026-8925 .- 1432-1874. ; 261:6, s. 967-976
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex.
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| 2. |
- Friant, S, et al.
(författare)
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Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(iMet) primer are required for initiation of reverse transcription in vivo
- 1998
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 18:2, s. 799-806
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Tidskriftsartikel (refereegranskat)abstract
- Reverse transcription of the Saccharomyces cerevisiae Ty1 retrotransposon is primed by tRNA(iMet) base paired to the primer binding site (PBS) near the 5' end of Ty1 genomic RNA. The 10-nucleotide PBS is complementary to the last 10 nucleotides of the acceptor stem of tRNA(iMet). A structural probing study of the interactions between the Ty1 RNA template and the tRNA(iMet) primer showed that besides interactions between the PBS and the 3' end of tRNA(iMet), three short regions of Ty1 RNA, named boxes 0, 1, and 2.1, interact with the T and D stems and loops of tRNA(iMet). To determine if these sequences are important for the reverse transcription pathway of the Ty1 retrotransposon, mutant Ty1 elements and tRNA(iMet) were tested for the ability to support transposition. We show that the Ty1 boxes and the complementary sequences in the T and D stems and loops of tRNA(iMet) contain bases that are critical for Ty1 retrotransposition. Disruption of 1 or 2 bp between tRNA(iMet) and box 0, 1, or 2.1 dramatically decreases the level of transposition. Compensatory mutations which restore base pairing between the primer and the template restore transposition. Analysis of the reverse transcription intermediates generated inside Ty1 virus-like particles indicates that initiation of minus-strand strong-stop DNA synthesis is affected by mutations disrupting complementarity between Ty1 RNA and primer tRNA(iMet).
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