RNA binding activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit from Chlamydomonas reinhardtii.
Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289-16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5'-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one- and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNA-binding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.
Yosef
Ido
I
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Irihimovitch
Vered
V
Knopf
Joel A
JA
Cohen
Idan
I
Orr-Dahan
Irit
I
Nahum
Eyal
E
Keasar
Chen
C
Shapira
Michal
M
D020121:5' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002736:Chloroplasts / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione; D019803:Glutathione Disulfide / Q000494:pharmacology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000737:chemistry; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature; D014466:Ultraviolet Rays
D020121:5' Untranslated Regions; D003432:Cross-Linking Reagents; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012313:RNA; D012273:Ribulose-Bisphosphate Carboxylase; D005978:Glutathione; D010100:Oxygen; D019803:Glutathione Disulfide