טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGiloh Mordechay
SubjectThe Enzyme RNase II and its Role in Degradation of RNA in
Bacteria and Plants
DepartmentDepartment of Biology
Supervisor Professor Gadi Schuster


Abstract

Exoribonucleases that degrade RNA molecules in the 3’ to 5’ direction are key enzymes in RNA processing and degradation. In this work we study Ribonuclease II, a hydrolytic 3’ to 5’ exoribonuclease that in earlier studies has been considered as mainly redundant to polynucleotide phosphorylase (PNPase). We used as a model the enzyme from the cyanobacterium Synechocystis sp. PCC 6803. RNase II belongs to a superfamily of proteins that are similar to Ribonuclease R, an E.coli paralogue to RNase II and is widespread in prokaryotic and eukaryotic organisms. We found that the prokaryotic versions can be classified into 3 main classes that are phylogenetically distinguishable. The importance of a well-conserved motif was investigated. Recombinant RNase II possesses nucleolytic activity, which requires magnesium and is dependent on nucleotide monophosphates, a previously unknown cofactor. In contrast with PNPase the protein had no preference for poly(A), but high binding affinity to poly(G). RNA stem-loop structures were relatively stable.  The regulation of RNase II and PNPase was further examined and compared with endonuclease in extracts from Synechocystis. The ratio of hydrolase to phosphorylase varied from 3 to 0.5, depending on magnesium concentration. Endonuclease dominated at low magnesium concentrations. Paradoxically, increasing concentrations stabilized the RNA while activating exonuclases. Hydrolase and phosphorylase responded differently to increasing magnesium concentrations. In extracts from dark-grown cells the relative contribution of hydrolase was diminished. The results of this work suggest a role of RNase II in widening the range of physiological conditions where RNA degradation is efficient. The existence of a novel RNA degradation mechanism in bacteria and chloroplasts without preceding polyadenylation, where RNase II is the main player, may also be implied.