D K Morris1, V Lundblad. 1. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Abstract
BACKGROUND: Telomeres are replicated in most eukaryotes by the enzyme telomerase, a specialized reverse transcriptase. A genetic screen in Saccharomyces cerevisiae designed to detect telomerase components previously led to the identification of four EST ('ever shorter telomeres') genes which are required for telomerase function in vivo. This report describes the cloning and characterization of EST3. RESULTS: We identified a potential site of +1 ribosomal frameshifting in the EST3 coding sequence and demonstrated that translation both upstream and downstream of this site is required for EST3 function. Mutation of EST3 such that it could not frameshift resulted in a strain with the same phenotype as an est3 null mutant, showing that EST3 frameshifting is required for telomere replication. Immunoblot analysis revealed that two proteins were synthesized from EST3: a truncated protein resulting from translation of only the first open reading frame, as well as the full-length 181 amino-acid Est3 protein resulting from translation through the frameshift site. Only the full-length Est3 protein was required for normal EST3 function. CONCLUSIONS: A programmed translational frameshifting mechanism similar to that used by yeast retrotransposons is employed to produce full-length Est3 protein. This is the first example in yeast of a cellular gene that uses frameshifting to make its protein product, and a potential link is suggested between retrotransposition and the telomerase pathway for telomere maintenance.
BACKGROUND: Telomeres are replicated in most eukaryotes by the enzyme telomerase, a specialized reverse transcriptase. A genetic screen in Saccharomyces cerevisiae designed to detect telomerase components previously led to the identification of four EST ('ever shorter telomeres') genes which are required for telomerase function in vivo. This report describes the cloning and characterization of EST3. RESULTS: We identified a potential site of +1 ribosomal frameshifting in the EST3 coding sequence and demonstrated that translation both upstream and downstream of this site is required for EST3 function. Mutation of EST3 such that it could not frameshift resulted in a strain with the same phenotype as an est3 null mutant, showing that EST3 frameshifting is required for telomere replication. Immunoblot analysis revealed that two proteins were synthesized from EST3: a truncated protein resulting from translation of only the first open reading frame, as well as the full-length 181 amino-acid Est3 protein resulting from translation through the frameshift site. Only the full-length Est3 protein was required for normal EST3 function. CONCLUSIONS: A programmed translational frameshifting mechanism similar to that used by yeast retrotransposons is employed to produce full-length Est3 protein. This is the first example in yeast of a cellular gene that uses frameshifting to make its protein product, and a potential link is suggested between retrotransposition and the telomerase pathway for telomere maintenance.