Defining the functional surface of the Est2 catalytic subunit of yeast telomerase
- Author(s): Nguyen, Lisa
- et al.
Telomerase is a ribonucleoprotein complex important for elongating ends of chromosomes. The catalytic subunit of telomerase (called Est2 in budding yeast) is made up of multiple domains. Here, I used a genetic strategy for defining the functional surface of Est2. A reverse- mutagenesis strategy that targets specific amino acids was used to select for candidate mutations. These mutations were then screened for effects on telomere replication in an over-expression dominant negative (ODN) and loss-of- function (LOF) assay. This strategy has generated a collection of separation-of-function (sof) alleles in each domain that are proposed to be located on the surface of the Est2 protein. Subsequent biochemical analysis has shown that the amino-terminal (TEN) domain of Est2 plays a direct or indirect physical interaction with Est3 and that multiple regions (TEN, TRBD and RT domains) of Est2 have an RNA binding function