Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA.

摘要: Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation telomerase often associated with immortalization human cells and cancer. To dissect enzyme mechanism, we developed functional vitro reconstitution assay. After removal essential 445 nucleotide RNA (hTR) by micrococcal nuclease digestion partially purified telomerase, transcribed hTR reconstituted activity. The activity was dependent upon specific to hTR. Using this assay, truncations at 5' 3' ends identified region hTR, similar size full-length RNAs from ciliates. This located between positions 1-203. Furthermore, found residues 1-44, template (residues 46-56) are not for activity, indicating minimal 44-203. Mutagenesis 170-179, 180-189 or 190-199 almost completely abolished ability function suggesting sequences structures within 30 required perhaps binding protein components.