Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunit in Vivo.

ABSTRACT: A leading objective in biology is to identify the complete set of activities that each gene performs in vivo In this study, we have asked whether a genetic approach can provide an efficient means of achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function (sof^-) mutations in a gene. Toward this goal, we have subjected the Saccharomyces cerevisiae EST1 gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of sof^- mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an est1-∆ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of sof^- mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell.

SUBMITTER: Lubin JW 

PROVIDER: S-EPMC5753878 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature