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P Farabaugh

Publications and source records attributed to P Farabaugh.

4 recordsLinked to original sources

Enhancer and silencerlike sites within the transcribed portion of a Ty2 transposable element of Saccharomyces cerevisiae.

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the element. This region had a dramatic effect, causing an approximately 1,000-fold increase in steady-state levels of RNA. The region stimulated transcription when placed in either orientation upstream of a heterologous gene, HIS4, lacking its own upstream activation sequence (UAS). We termed this positively acting region an enhancer, by analogy to sites described in higher cells, to distinguish it from yeast UASs which do not function when placed within the transcribed portion of the gene. Though, like some higher eucaryotic enhancers, the Ty2-917 enhancer is located within the transcribed region, it is unlike them in that it occurs within a coding region rather than in an intron. The Ty2-917 enhancer and the Ty2-917 UAS had a synergistic effect on transcription, together stimulating transcription 15-fold over the predicted additive effect. We also identified a site which decreases RNA accumulation, located about 750 base pairs into the element. This site functioned in only one orientation when inserted upstream of the UAS-less heterologous gene. The site was similar to silencers, or negative enhancers, in that it acted to repress transcription from outside the transcribed region, but was distinct in that the function of a canonical silencer was independent of orientation.

Base Sequence

Three genes are required for trans-activation of Ty transcription in yeast.

Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo delta insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty delta-delta transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for transcription initiation in delta sequences. We have selected for new spt mutations and have screened for those with the unique suppression pattern of spt3 mutations with respect to two insertion mutations. Our selection and screen has identified two additional genes, SPT7 and SPT8, that are also required for transcription initiation in delta sequences. We show that mutations in SPT7 or SPT8 result in the same alteration of Ty transcription as do mutations in SPT3. In addition, mutations in all three genes cause a sporulation defect. By assay of a Ty-lacZ fusion we have shown that spt3, spt7 and spt8 mutations reduce transcription from a delta sequence by 10-25-fold. Finally, we show that SPT3 mRNA levels are unaffected in either spt7 or spt8 mutants, suggesting that these two genes do not regulate transcription of SPT3.

DNA Transposable Elements

Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression.

We have determined the DNA sequence of the transposable element Ty912 of yeast. The 5918-base-pair element encodes two genes, tya912 and tyb912, which specify proteins similar to sequence-specific DNA-binding proteins of Escherichia coli and retroviral reverse transcriptases, respectively. The tyb912 gene is atypical of eukaryotic genes since (i) it begins 1336 nucleotides into the Ty912 mRNA (i.e., downstream of the tya912 gene) and (ii) the first in-frame AUG is 921 nucleotides into the coding frame. Protein blot analysis of Ty-lacZ fusions shows that the tyb912 gene is translated starting at the 5' end of the tya912 gene and that the primary translational product is a tya912::tyb912 fusion protein. We have shown that synthesis of this fusion protein probably does not occur by RNA splicing. The data are consistent with a mechanism of translational frameshifting occurring within the region of overlap between the 3' end of tya912 and the 5' end of tyb912.

Amino Acid Sequence