Expression and Purification of Zinc Finger Domain and Central Domain of MDMD2

[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and preliminarily identified biologic activity of the purified fusion proteins. [ Method ] ZF and CT cod- ing regions were amplified from MDM2 cDNA library by PCR and separately inserted into prokaryotie expression vector pET28b to construct the recombinant plas- mids. After verification by enzyme digestion, the recombinant plasmids were separately transformed into E. coli DH5c~ competent cells. The expressed recombinant plasmids were purified using Ni-NTA magnetic beads and identified by SDS-PAGE and Western blotting. [ Result] The molecular weight of His-ZF and His-CT fu- sion proteins was 21 and 31 kD, respectively. E Conclusion] Recombinant fusion proteins containing ZF and CT of MDM2 were obtained successfully, which laid the foundation for subsequent protein crystallization and three-dimensional structure analysis.

Optimization of RNA Secondary Structure Prediction with SHAPE(Selective 2'-hydroxyl Acylation Analyzed by Primer Extension)Chemistry

SHAPE （selective T-hydroxyl acylation analyzed by primer extension） chemistry has been widely used in the prediction of RNA secondary structure at single-nucleotide solution. This study was designed to determine the optimal concentrations of RNA and modifying reagent in SHAPE analysis to improve the accura- cy of structure prediction. The results showed that at the ratio of N-methylisatuic anhydride （NMIA） （μmol/L） to RNA （0μmol/L） of 13： 20, the resulting concen- tration of full-length RNA was the highest, and the sequencing signal was the strongest. So, this ratio is optimal for the analysis and prediction of RNA secondary structure in SHAPE chemistry.

Prediction of the Structure of Full-length Human Telomer-ase RNA with SHAPE(Selective 2'-hydroxyl Acylation An-alyzed by Primer Extension)

Telomerase is a unique ribonucleoprotein complex that protects chromosomes from degradation and recombination. The defects in telomerase activity eansed by the mutations in telomerase RNA （hTER） is associated with several human genetic diseases, so the molecular biology and structural biology of hTER are of great significance for the treatment of related diseases and the development of corresponding drugs. In this study, hTER was obtained by in vitro transcription, and then modified with N-methylisatoic anhydride （NMIA） by SHAPE （selective 2＇-hydroxyl aeylation analyzed by primer extension） ; finally, the eDNA fragment was obtained by reverse transcription to analyze the free nucleotides and dynamic structure of hTER, which may provide experimental basis for analyzing three--di- mensional structure of hTER obtained in vitro. In addition, the SHAPE parameters for long RNA fragment （ 〉 300 nt） were optimized. The results showed that 1 μmol/L hTER and 3.9 mmol/L NMIA were optimal, and 49 ℃ was the best temperature for primer extension.