C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.

A yeast protein with homology to the beta-subunit of G proteins is involved in control of heme-regulated and catabolite-repressed genes.

The product of the Saccharomyces cerevisiae AER2 gene is responsible for maintaining repression of at least two distinct regulatory pathways: heme activation/repression and catabolite repression. Mutations in the gene caused an eightfold increase in the expression of the heme-activated CYC1 gene in the absence of heme, a substantial increase in the expression of the heme-repressed ANB1 gene in the presence of heme, and a 13-fold increase in the expression of the catabolite-repressed GAL1 gene in the presence of glucose. Lesser or no increases in the expression of these genes were observed under derepressed or activation conditions. The aer2 mutations also caused a large increase in CYC7 gene expression under all conditions; this gene is subject to heme activation/repression, as well as catabolite repression. The AER2 gene was cloned and the sequence determined. The large open reading frame contiguous with the transcript from the complementing region encoded a 713-amino acid polypeptide chain with extensive homology to the beta-subunit of G proteins. The sequence revealed that AER2 is the TUP1 gene. A deletion mutation was constructed and the null phenotype was the same as the original mutants. The aer2 null mutant was shown to have increased aerobic and anaerobic levels of RNA encoding the ROX1 repressor, normally expressed only aerobically and responsible for the aerobic repression of ANB1 expression. The increase in both ROX1 and ANB1 RNAs aerobically in this mutant suggests that the repressor is nonfunctional in the mutant.