The core of the telomerase enzyme is the TERT and TER subunits. In budding yeast, the EST2 gene encodes the protein subunit while the TLC1 gene encodes the telomerase RNA (Lingner et al., 1997; Singer and Gottschling, 1994). The EST2 gene name stands for Ever Shorter Telomeres 2, because the telomere repeat tracts in these mutants that have inactivated the EST2 gene continuously shorten until the most of the cells in the culture senesce. Four EST genes were isolated in the screen that identified EST2, yielding the additional protein-encoding genes EST1, EST3 and EST4 (EST4 was found to be the previously characterized CDC13 gene, and the EST4 mutant is commonly referred to as cdc13-2 or cdc13est) (Lendvay et al., 1996). The mutants isolated in these genes all show the same phenotype, indicating a loss of telomerase activity in vivo. However, cell extracts from est1, est3 or cdc13est mutants all yield telomerase activity in vitro (Lingner et al., 1997). Elegant work has since demonstrated that the EST1, EST3 and CDC13 gene, products serve as adaptor proteins to help recruit the telomerase enzyme to the telomere, and the molecular dynamics of this process is under intensive study by many groups (Evans and Lundblad, 1999; Smith et al., 2003; Taggart et al., 2002). One EST1 paralog has been identified in budding yeast. Two orthologs in fission yeast and three EST1 orthologs in human cells have been identified, and some of these proteins associate with telomerase and telomerase RNA (Lundblad, 2003; Sanger_Institute 2005; Zhou et al., 2000). While CDC13 has no ortholog beyond budding yeasts, it has a similar structure compared to the telomere end-binding factor POT1 in S. pombe and mammals.
Telomere structural proteins Double-stranded yeast telomere repeats are bound predominantly by Repressor Activator Protein 1 or RAP1 protein. This protein represses transcription when bound to the yeast silent mating type cassettes (described below) and telomeres, activates the transcription of many housekeeping genes, and serves as a crucial telomere length regulator and chromatin component. RAP1 protein contains an ~ 230 amino acid DNA binding domain and an ~170 amino acid C-terminal domain which serves as an interaction site for many telomere-associated proteins (Hardy et al., 1992; Henry et al., 1990; Konig et al., 1996; Moretti et al., 1994; Wotton and Shore, 1997). Thus, RAP1 encodes a multifunctional protein whose activities are dependent upon its DNA binding context. At the telomere, RAP1 protein is known for its association with the gene-silencing proteins SIR2, SIR3 and SIR4 and the telomere length regulatory proteins encoded by RIF1 and RIF2.
Gene silencing in budding yeast, a chromatin-mediated effect that prevents the transcription of genes, was first discovered in the late 1970s (Rine et al., 1979). When reporter genes are placed adjacent to telomere repeats in the chromosome, their expression is repressed or silenced. Similar types of telomere-associated silencing have been reported in S. pombe, Drosophila melanogaster and humans (Baur et al., 2001; Levis et al., 1985; Nimmo et al., 1994) in addition to other organisms. In budding yeast, silencing has been found to be effected by a number of chromatin proteins and chromatin modifying and remodeling complexes. The "classic" proteins involved are encoded by SIR2, SIR3 and SIR4. These proteins also act with the SIR1 protein to silence two specialized loci, the silent mating type cassettes, and SIR2 protein also acts with other proteins to repress RNA polymerase II transcription and recombination in the array of ribosomal RNA genes called the rDNA array (Figure 17.2).
Of SIR2, SIR3 and SIR4, only SIR2 has orthologs in a large number of other organisms. SIR2 encodes an NAD+-dependent deacetylase that is important in maintaining gene silencing and prolonging yeast lifespan (reviewed by Blander and Guarente (2004)). Many organisms encode multiple SIR2 family members, or sirtuins, and the suggestion has been made that sirtuins may alter protein posttranslational modification to regulate responses to stress that also affect lifespan (Blander and Guarente, 2004).
In budding yeast, the SIR proteins are one of the first examples of telomeres serving as a reservoir of proteins that can be mobilized to move to other genetic loci.
ACS RaP1 site
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