טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFridman Yearit
SubjectDevelopment of Methods for Selective Labeling of Telomerase
with Fluorophores for Analysis of Conformational
Changes by Fluorescence Resonance Energy
Transfer (FRET)
DepartmentDepartment of Biology
Supervisor Professor Haim Manor


Abstract

Telomeres are DNA - protein structures found at the ends of linear eukaryotic chromosomes. They protect the chromosomes from degradation and end to end fusion. The eukaryotic DNA replication apparatus cannot finish the replication of linear DNA molecules. Therefore, in many types of cells, the telomeres are shortened after each cell division. In other type of cells, particularly cancer cells, telomere shortening is prevented by the presence of the enzyme telomerase. 


Telomerase is a specialized reverse transcriptase. Unlike other reverse transcriptases, telomerase uses as a template an RNA template which is an integral part of the enzyme. In addition to the RNA, which is designated Telomerase RNA (TER), the enzyme contains a catalytic protein subunit, designated TERT (Telomerase Reverse Transcriptase), and auxiliary proteins.


The aim of my research was to study conformational changes that occur in the RNA component of the telomerase after its assembly with the protein subunit. I used for this study in vitro reconstituted Tetrahymena telomerase core consisting of TERT and TER, and the technique of Fluorescence Resonance Energy Transfer (FRET). To perform this study, I developed techniques for labeling the TER molecules at their 5'-end with a donor fluorophore and at their 3'-end with an acceptor fluorophore. I first measured the FRET that occurred between the two ends in purified TER molecules. Using these data, I calculated a distance of 45Å between the two ends. Next, I reconstituted the core enzyme with the labeled TER molecules and measured FRET again. The data indicated that the distance between the 5' and 3' ends of the TER molecules increased upon enzyme assembly, an indication of a significant conformational change that occurred in the RNA. This work lays the foundation for further studies on conformational changes that occur in telomerase during assembly and catalysis.