|M.Sc Student||Shira Herlich|
|Subject||Unravelling the Mechanism of Activity of the Chloroplast|
PNPase by Site Directed Mutagenesis
|Department||Department of Biology||Supervisor||Full Professor Schuster Gadi|
|Full Thesis text|
Polynucleotide phosphorylase (PNPase) is a phosphorolytic exoribonuclease that plays an important role in degradation, processing, and polyadenylation of RNA in prokaryotes and organelles. Systematic structure-function analysis of chloroplast PNPase has been so far very limited, and its roles in plant organelles are poorly understood. In order to study it we have used in vitro mutagenesis followed by functional analysis.
Our initial goal was to express the chloroplast PNPase (cpPNPase) from Arabidopsis thaliana in bacteria, and characterize its activities in vitro. The results obtained for the Arabidopsis PNPase were similar to the previously characterized bacterial and spinach chloroplast PNPase.
Following the analysis of the recombinant protein, we aimed to achieve the main goal of this work: explore the domains of cpPNPase and identify potential areas that affect its various activities. PNPase in its monomeric form consists of two RNase PH-like segments, known as core domains, as well as KH and S1 RNA-binding domains. Using site directed mutagenesis, we created the G596R, P184L and P184L-R176S mutated versions of the protein. Each mutant was analyzed for its binding, degrading, and polyadenylating RNA transcripts. Only G596R was located at the phosphorolytic active site in the second core domain, and indeed this mutation abolished all activities and binding capability, as well as failure to create a distinct oligomeric structure. Although the first core domain does not harbor the catalytic site, a double mutation in this area, R176S-P184L, located on the same α-helix, reduced RNA affinity and led to inability of the RNA to enter the phosphorolytic site, hence a complete inhibition of activity as witnessed. On the other hand, P184L alone displayed similar RNA affinity to the WT, but intermediate phosphorolytic activity between the WT and the inactive or null mutants. In this case, we hypothesized that the conformational change induced by our mutations still enabled RNA binding, and thus reduced but not eliminated enzyme activity. We considered that the first core domain mutations might have directly altered the oligomeric structure of the enzyme and its processivity, however they still formed hexamers akin to the WT.
Taken together, these results confirm a structural role for the first core domain of PNPase in the modulation of the observed catalytic activity even though the catalytic site is located in the second core domain.