טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLitovco Phyana
SubjectCharacterization of a Multimeric Complex of the Proteins
Translin and TRAX from Schizosaccharomyces Pombe
DepartmentDepartment of Biology
Supervisors Professor Haim Manor
Professor Yoav Arava
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Translin was identified in extracts from human leukemia and lymphoma cells as a protein that bound single-stranded DNA consisting of consensus sequences found next to chromosomal translocations. It was, therefore, proposed that it is involved in chromosomal translocations. Our laboratory showed that recombinant human Translin binds d(GT)n repeats and telomeric d(TTAGGG)n repeats with much higher affinities. Hence, we suggested that human Translin might be involved in the metabolism of d(GT)n?(AC)n microsatellites and human telomeres. Based on other studies, which indicated that Translin also binds specific RNA sequences, it has been suggested that it is involved in transport and translation of specific mRNAs. TRAX, a Translin paralogue, was identified by Yeast Two-Hybrid assays as a protein which forms specific complexes with Translin. Both Translin and TRAX were subsequently found to be highly conserved in eukaryotes. More recent studies provided evidence for the involvement of Translin-TRAX complexes in RNA interference. The fission yeast S.pombe was chosen by our laboratory as a model system for characterizing Translin and TRAX functions, because of its rapid cell proliferation, ease of maintenance and the possibility of genetic manipulation. We began these studies by cloning the open reading frames encoding Translin and TRAX from a library of S.pombe cDNA. Then we found that the two proteins are actually expressed in S.pombe cells and that neither is essential for S.pombe cell proliferation. Subsequently, we mapped the interaction surface of spTranslin and found that it resides in the equatorial part of the protein. We also mapped protein-protein interaction surfaces of spTranslin at regions including its carboxy- or amino- termini. In my research, I cloned the ORFs encoding the S.pombe Translin and TRAX in a single expression plasmid that has been transfected into E.coli cells. Then, I purified from these cells Translin-TRAX complexes and showed that, like pure Translin from S.pombe, these complexes have a higher affinity towards single-stranded RNA than towards single-stranded DNA. I also carried out a series of glycerol gradient sedimentation analyses of the S.pombe Translin-TRAX complexes and found that they include two major populations: tetramers and octamers. Further analysis indicated that these two populations are in equilibrium. Addition of RNA to the mixtures of the two proteins shifts the equilibrium towards formation of octamers bound to RNA. Taken together, my data suggest that the octamer configuration is the functional form of both pure Translin and Translin-TRAX complexes.