Characterization of recombinant Saccharomyces cerevisiae telomerase core enzyme purified from yeast.
Telomerase is a cellular reverse transcriptase that elongates the single-stranded chromosome ends and oligonucleotides in vivo and in vitro. In Saccharomyces cerevisiae, Est2p (telomerase catalytic subunit) and Tlc1 (telomerase RNA template subunit) constitute the telomerase core complex. We co-overexpressed GST (glutathione S-transferase)-Est2p and Tlc1 in S. cerevisiae, and reconstituted the telomerase activity. The GST-Est2p-Tlc1 complex was partially purified by ammonium sulphate fractionation and affinity chromatography on glutathione beads, and the partially purified telomerase did not contain the other two subunits of the telomerase holoenzyme, Est1p and Est3p. The purified recombinant GST-Est2p-Tlc1 telomerase core complex could specifically add nucleotides on to the single-stranded TG(1-3) primer in a processive manner, but could not translocate to synthesize more than one telomeric repeat. The purified telomerase core complex exhibited different activities when primers were paired with the Tlc1 template at different positions. The procedure of reconstitution and purification of telomerase core enzyme that we have developed now allows for further mechanistic studies of the functions of other subunits of the telomerase holoenzyme as well as other telomerase regulation proteins.
Liao
Xin-Hua
XH
Max-Planck Junior Research Group in the State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
Zhang
Ming-Liang
ML
Yang
Cui-Ping
CP
Xu
Lu-Xia
LX
Zhou
Jin-Qiu
JQ
D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012965:Sodium Chloride; D019098:Telomerase / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D012965:Sodium Chloride; D019098:Telomerase