Poster: Oxidative stress

Abs # 67: RNA Binding Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii

Presenter:	Knopf, Joel A., knopfj@bgumail.bgu.ac.il
Authors	Knopf, Joel A. (A)   Yosef, Ido  (A)   Irihimovitz, Vered  (A)   Cohen, Idan  (A)   Shapira, Michal  (A)
Affiliations:	(A): Ben Gurion University of the Negev Department of Life Sciences

Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generates an oxidative stress that leads to a dramatic arrest in the synthesis of the large subunit (LSU) of Rubisco. The translational arrest correlates with transient changes in the intracellular levels of reactive oxygen species and with the shifting of the glutathione pool towards its oxidized form. We examined how the redox potential of glutathione affects the RNA-protein interactions with the 5' UTR of rbcL. This RNA region specifically binds a group of proteins with molecular weights of 81, 62, 51 and 47 kDa in UV cross-linking experiments. Binding under oxidizing conditions gave a different pattern, and a new protein of 55kDa was shown to interact with the RNA. The 55 kDa protein co-migrated with Rubisco LSU on one-and two- dimensional gels, and its RNA-binding activity was further verified by using the purified protein in UV cross-linking experiments. However, this binding was found to be sequence-independent. The purified Rubisco bound RNA with an apparent Kd of 9.9 nM. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU and the RNA binding domain (RBD), a known motif among RNA binding proteins. A recombinant polypeptide, corresponding to the amino-terminal domain of the LSU, bound RNA in vitro, with an apparent Kd of 7.9 nM. An oxidizing environment was required for RNA binding only with the complete LSU, while the amino terminus bound RNA under all conditions. No RNA binding activity was found in the carboxy-terminal domain of LSU. The crystallographic structure of Rubisco indicates that the amino terminus is buried within the LSU. We suggest that under oxidizing conditions this domain is exposed, enabling its interaction with RNA.