Modeling and structure function analysis of the putative anchor site of yeast telomerase.
Telomerase is a ribonucleoprotein reverse transcriptase responsible for extending one strand of the telomere terminal repeats. Unique among reverse transcriptases, telomerase is thought to possess a DNA-binding domain (known as anchor site) that allows the enzyme to add telomere repeats processively. Previous crosslinking and mutagenesis studies have mapped the anchor site to an N-terminal region of TERT, and the structure of this region of Tetrahymena TERT was recently determined at atomic resolutions. Here we use a combination of homology modeling, electrostatic calculation and site-specific mutagenesis analysis to identify a positively charged, functionally important surface patch on yeast TERT. This patch is lined by both conserved and non-conserved residues, which when mutated, caused loss of telomerase processivity in vitro and telomere shortening in vivo. In addition, we demonstrate that a point mutation in this domain of yeast TERT simultaneously enhanced the repeat addition processivity of telomerase and caused telomere elongation. Our data argue that telomerase anchor site has evolved species-specific residues to interact with species-specific telomere repeats. The data also reinforce the importance of telomerase processivity in regulating telomere length.
Lue
Neal F
NF
Department of Microbiology & Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. nflue@med.cornell.edu
Li
Zhaohui
Z
D000818:Animals; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016615:Telomere / Q000378:metabolism; D013768:Tetrahymena / Q000201:enzymology
D029701:Saccharomyces cerevisiae Proteins; C422952:telomerase RNA; D012313:RNA; C509185:EST2 protein, S cerevisiae; D019098:Telomerase