טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEliahoo Elad
SubjectStudies on the Structure and Function of the Protein
Translin in Schizosaccharomyces Pombe
DepartmentDepartment of Biology
Supervisors Professor Haim Manor
Professor Amnon Harel
Full Thesis textFull thesis text - English Version


Abstract

Translin is a single-stranded RNA- and DNA-binding protein, which has been highly conserved in eukaryotes. TRAX is a Translin paralog associated with Translin.

We studied the structure of Schizosaccharomyces pombe Translin (spTranslin) using a combined bioinformatics and molecular genetics approach, as follows: We generated structural models of spTranslin based on the solved 3D structure of the human ortholog. Using bioinformatics, we identified in the equatorial part of the protein a putative nucleic acids interaction surface, which includes polar and positively charged residues surrounding a shallow cavity. Experimental verification of the bioinformatics predictions was obtained by assays of nucleic acids binding to amino acid substitution variants. In addition, bioinformatics combined with yeast two-hybrid assays and proteomic analyses identified at the C-terminus of the spTranslin structure a region required for interaction with spTRAX and for spTranslin dimerization. Bioinformatics also predicted the presence of a second protein-protein interaction site at the N-terminus of the structure. Finally, similar nucleic acids and protein interaction sites were predicted for the human Translin. Thus, our results appear to generally apply to the Translin family of proteins.

In the second part of this work, we used proteomic techniques to elucidate the functions of spTranslin. We performed screening by affinity purification of complexes including spTranslin from S. pombe cells expressing either tagged or untagged spTranslin and identification of the proteins in these complexes by mass spectrometry.  To distinguish between specifically associated proteins from false positives, we used differential labeling of spTranslin expressing cells and control cells with nitrogen heavy isotopes. Mass spectrometry and statistical techniques were utilized to identify proteins having significantly high (or low) 15N /14N ratios. These experiments led to identification of three proteins, of which two, named Srp1 and GAPDH, associate with spTranslin in the presence of RNA and one, named vip1, whose association with spTranslin is independent of the presence of RNA.

Finally, we report, for the first time, that cells from which the spTranslin encoding gene has been deleted are hypersensitive to the ribosomal antibiotic Anisomycin. 

Taken together, our data support the involvement of complexes including spTranslin, spTRAX, Srp1 and GAPDH, in regulation of ribosomal precursors transport and in ribosome function. Another complex including spTranslin, spTRAX, GAPDH and vip1 could be involved in transport of specific mRNAs.