Preparation And Physical And Chemical Properties Study Of Functional Domain Of Yeast Telomerase RNA

Telomerase is a catalytic ribo-nucleoprotein reverse transcriptase, consisting of threesubunits. TR and TERT are key components for catalyzing: TR provides the template, TERTsynthesizes telomeric DNA repeats addition to chromosome ends, which prevents fromtelomere shortening during subsequent cell division. Shortened telomere will lead to thelimitation of replication, and reduce the multiplication capacity in cell. Telomerase activityhas been strictly regulated in normal somatic cells, and has only been detected in some vitalcells, including hematopoietic stem cells, germ cells and stem cells. Telomerase activity hasbeen inhibited in normal somatic cells but reactivated in tumor cells leading to transformationof malignant tumor. Telomerase plays an important role in maintaining the stability andintegrity of chromosome ends DNA.Yeast telomerase is an ideal research model. In order to investigate the structure andmolecule mechanism in yeast telomerase, we focused on the central activity region of yeastTR which ultimately aimed to form complex with catalytic subunit TERT for telomerasestructure and mechanism research. First of all, we purified T7RNA polymerase, and thenprepared yeast telomerase RNA functional region with high purity by ribozyme HH and HDVself-cleavage effect, at the meantime studied the physical factors affecting yeast TR stabilityand correct folding in different conditions. Our results would provide biological material basisfor the research on telomerase RNA structure，and lay the foundation of intensive study oftelomerase. The main results are as follows:（1） We explored a quick method for purification of T7RNA polymerase: Tandom SP-FFand DEAE-FF columns method, using strong cation exchange column SP and weak anionexchange column DEAE to purify T7RNA polymerase fast and effectively; The purified T7RNA polymerase had high enzymetic activity, and the transcribed RNA showed no obviousdegradation.（2） Using restriction enzymes PstI and NgoMIV to digest plasmids HH-Yeast–pES andpSP72-Cabullse-HDV, and conduct ligation of the PstI-Yeast-HH-239-NgoMIV fragment intothe vector pSP72-Cabullse-HDV （PstI/NgoMIV）, the final reconstructed plasmidpSP72-HH-Yeast-HDV-239with T7promoter, ribozyme HH, ribozyme HDV andYeast-RNA-239was obtained.PCR amplification of EcoRI-Yeast-HDV-239-XhoI andEcoRI-Yeast-239-XhoI gene fragments with pSP72-HH-Yeast-HDV-239as the template,using EcoRI and XhoI to digest plasmid pSP72-Cabullse-HDV andEcoRI-Yeast-HDV-239-XhoI and EcoRI-Yeast-239-XhoI gene fragments, and to ligase thefragments and the vector, reconstructed plasmids pSP72-Yeast-HDV-239andpSP72-Yeast-239were obtained with T7promoter、ribozyme HDV and Yeast-RNA-239andT7promoter and Yeast-RNA-239respectively.（3） Performing in vitro transcription using XhoI linearized reconstructed plasmidpSP72-Yeast-HDV-239. Molecule sieve chromatography SuperDeX-200was utilized toseperate and purify Yeast-RNA-239, Yeast TR Yeast-RNA-239was acquired with high homogeneity.（4） Yeast TR Yeast-RNA-239was dissolved in buffers containing different concentrationof Na⁺and Mg²⁺, yeast TR showed two conformations as the metal ions concentrationincreased. This result proved that Na⁺and Mg²⁺ concentration had some impact onconformation of yeast TR.（5） Yeast TR Yeast-RNA-239formed a more and more homogeneous and compactcomplex with Tetrahymena thermophila telomerase-associated protein p65as increased p65concentration. This result suggested that p65could combine yeast TR effectively.