Alistair Chambers

In Vivo Analysis of Functional Regions within Yeast Rap1p

ABSTRACT We have analyzed the in vivo importance of different regions of Rap1p, a yeast transcriptional regulator and telomere binding protein. A yeast strain (SCR101) containing a regulatable RAP1 gene was used to test functional complementation by a range of Rap1p derivatives. These experiments demonstrated that the C terminus of the protein, containing the putative transcriptional activation domain and the regions involved in silencing and telomere function, is not absolutely essential for cell growth, a result confirmed by sporulation of a diploid strain containing a C terminal deletion derivative ofRAP1. Northern analysis with cells that expressed Rap1p lacking the transcriptional activation domain revealed that this region is important for the expression of only a subset of Rap1p-activated genes. The one essential region within Rap1p is the DNA binding domain. We have investigated the possibility that this region has additional functions. It contains two Myb-like subdomains separated by a linker region. Individual point mutations in the linker region had no effect on Rap1p function, although deletion of the region abolished cell growth. The second Myb-like subdomain contains a large unstructured loop of unknown function. Domain swap experiments with combinations of elements from DNA binding domains of Rap1p homologues from different yeasts revealed that major changes can be made to the amino acid composition of this region without affecting Rap1p function.

The multifunctional transcription factors Abf1p, Rap1p and Reb1p are required for full transcriptional activation of the chromosomalPGK gene inSaccharomyces cerevisiae

We have identified two new transcription factor binding sites upstream of the previously defined UAS within the phosphoglycerate kinase (PGK) gene promoter inSaccharomyces cerevisiae. These sites are bound in vitro by the multifunctional factors Cpf1p and Reb1p. We have generated targeted deletions of Rap1p, Abf1p and Reb1p binding sites in the promoter of the chromosomal copy of thePGK gene. Northern blot analysis confirmed that mostPGK promoter activity is mediated through the Rap1p binding site. However, significant effects are also mediated through both the Reb1p and Abf1p sites. In contrast, when the promoter is present on a high-copy-number plasmid, both the Abf1p and Reb1p sites play no role in transcriptional activation. The role of Cpf1p was examined using acfp1 null strain. Cpf1p was found to have little if any, effect on activation of either the chromosomal or plasmid-bornePGK gene.

A Reb1p‐binding site is required for efficient activation of the yeast RAP1 gene, but multiple binding sites for Rap1p are not essential

The Saccharomyces cersvislae RAP1 protein (Rap1p) is a key multifunctional transcription factor. Using gel retardation analysis, four binding sites for RAP1p have been identified within the promoter of the RAP1 gene. These sites are located downstream of a binding site for the transcription factor Reb1p. The Reb1p site and an associated AT‐rich region are important for transcriptional activation, but deletion of three of the RAP1 p‐binding sites had little effect on promoter activity. The activity of the RAP1 promoter has been analysed in a yeast strain (YDS410) that contains a temperature‐sensitive mutation In the RAP1 gene. This mutation renders the DNA‐binding activity of Rapip temperature dependent. When YDS410 was grown at a semi‐permissive temperature (30°C), the activity of the RAP1 promoter increased by approximately 170%, compared with the same strain grown at the permissive temperature (25°C). A RAP1 promoter in which three of the four RAP1 p‐binding sites had been deleted, showed only a small increase in activity in the same experiment. These data confirm that Rap1p is not required for activation of the RAP1 gene, and suggest a role for Rap1p In negative auto‐regulation.

Role of the N-terminal region of Rap1p in the transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

In the yeast two‐hybrid system, the N‐terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two‐hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N‐terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N‐terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N‐terminal region of Rap1p. These results suggest that one function of the N‐terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein–protein interaction. Copyright

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast (Saccharomyces cerevisiae) Rap1p has been expressed in fission yeast (Schizosaccharo‐myces pombe) under the control of the regulatable fructose bisphosphatase (fbp) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature‐sensitive mutation in the DNA‐binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1pts is severely inhibited. Removal of a short region downstream of the DNA‐binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.

Rap1p is a negative regulator of the RAP1 gene

Abstract The promoter of the RAP1 gene contains four potential binding sites for Rap1p, located between the UAS and the RNA initiation site. We have confirmed that three of these sites are recognised by Rap1p in vitro. Different combinations of the three sites were then mutated to abolish Rap1p binding, and the effect of these mutations on promoter activity was determined. When all three Rap1p sites were mutated, the activity of the promoter increased by about 130%, indicating that at least one of the sites is a negative element. Analysis of promoters with different combinations of the mutant sites revealed that the 5′-most site (A) is the principal target for repression. To test the involvement of Rap1p in controlling RAP1 expression, we have measured transcription of the chromosomal RAP1 gene in a RAP1 wild-type strain and two strains containing rap1ts mutations. At a semi-permissive temperature, the RAP1 promoter was more active in the rap1ts strains than in the RAP1 wild-type strain, suggesting that expression of the chromosomal RAP1 gene is greater when the activity of Rap1p in the cell is compromised. The activities of the wild-type promoter, and the promoter with mutations in the three Rap1p-binding sites, were compared in sir1, sir2, sir3 and sir4 mutant strains. In each case, the mutated promoter was significantly more active than the wild-type promoter, implying that the repression mechanism is not dependent on any one of the SIR gene products.