טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMouna Habib
SubjectIdentification and Characterization of the Ribonucleolytic
Activity of P53
DepartmentDepartment of Biology
Supervisor Full Professor Schuster Gadi
Full Thesis textFull thesis text - English Version


Abstract

The p53 tumor suppressor protein is a sequence-specific transcription factor which is important for cell growth, division and cell cycle. Most tumors in cancer sickness are characterized with mutations or changes in its expression. This leads to the altered regulation of hundreds of genes that are directly influenced by this TF. p53 is composed of multiple domains. It acts as a nuclear TF, when bound to DNA and assembles into a tetramer which is capable of recruiting other proteins to form a transactivation response on p53-target genes. In addition, it was shown that p53, in addition to binding RNA and ssDNA in sequence-independent manner, degrades ssDNA as a 3’ to 5’ exonuclease. This suggests the possible direct involvement of the p53 protein in DNA damage and repair response by forcing the cell to make a decision between survival and death. Followed this observation, as well as several results that p53 is possibly active as a ribonuclease degrading RNA, we asked whether a recombinant p53 is a ribonuclease. To this end the recombinant p53 protein, composed on the DNA binding and the tetramerization domains (rp53CT) was produced in E. coli and purified.  Using this recombinant protein, the excision of nucleotides from defined synthetic RNA substrates was probed.

Our results indicate that: (1) rp53CT is an endoribonuclease, capable of degrading ssRNA substrates. (2) rp53CT’s endonucleolytic activity is sensitive to secondary structures and dsRNA substrates. (3) Introducing point mutations at residues in the DNA binding site significantly inhibit its ribonuclease activity. (4) Histidine 115 was found to be important for both DNA and RNA degradation activities, suggesting the active sites for these activities are identical or overlapping. (5) Monomeric rp53CT is the ribonucleolyticly active form of the protein, while the tetramer form is devoid of this activity. Our results suggest a new characterized mode of rp53CT’s ribonucleolytic activity, and emphasises the importance of further studying and characterizing this activity and how it is related to the other previously known functions of the protein.