טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSheffy Levin Sharon
SubjectRNase E of Cyanobacteria and Higher Plants: A Key Enzyme
in RNA Metabolism
DepartmentDepartment of Biology
Supervisor Professor Gadi Schuster


Abstract

mRNA degradation in E. coli starts with an endonucleolitic cleavage of the target molecule by the enzyme Ribonuclease E (RNase E). Homologous genes of RNase E were detected in many prokaryotes, and lately also in the nuclear genome or ESTs of higher plants. The evolutionary origin of the chloroplast was an endosymbiotic event involving a eukaryotic cell and photosynthetic bacteria. The prokaryotic origin of RNase E led us to hypothesize that RNase E homologs in higher plants participate in mRNA degradation in the chloroplast. This key player in mRNA degradation, though extensively investigated in E. coli, has not been characterized yet in other organisms. Focusing on two organisms: Thermosynechococcus vulcanus, a thermophylic cyanobacteria, and Lycopersicon esculentum (Tomato), we studied the mechanism and factors controlling the activity of this enzyme as the assumed initiator of the mRNA degradation process.

The sequence of the rne gene from T. vulcanus was cloned, the protein was over-expressed in E. coli, and in vitro characterization revealed functional homology to E. coli RNase E.

The tomato rne cDNA was obtained by screening a cDNA library. Polyclonal anti-tomato RNase E antibodies were used to experimentally analyze the bioinformatic prediction that the tomato RNase E is targeted to the chloroplast. Tobacco plants over-expressing the tomato RNase E were generated, and molecular analysis of these, as well as Arabidopsis and tomato plants over-expressing this protein, will enable us to determine the cellular localization of this protein as well as its affect on RNA metabolism.