Abstract
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.
Highlights
When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS),1 that cause a variety of cellular and molecular damage
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)
We previously showed that transfer of the green algae C. reinhardtii from low light (70 mol mϪ2 sϪ1) to high light (700 mol mϪ2 sϪ1) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco [8]
Summary
Strains and Growth Conditions—C. reinhardtii wild type CC-125 cells were grown in high salt reduced sulfate medium with bubbling of 5% CO2 and constant rotary shaking at 25 °C. Samples from the heparin-Actigel column fractions (2 l containing approximately 9 g of protein) were preincubated for 10 min at room temperature with 5 units of RNasin (Promega) in 3 mM MgCl2 in a total volume of 5 l. Samples of proteins eluted from the heparinActigel column (2 l containing approximately 9 g of protein), purified Rubisco (25 ng), or the recombinant polypeptides that corresponded to Rubisco LSU (amino acids 1– 475) or its sub-fragments (amino acids 1–150 and 151– 475) after purification over a nickel nitrilotriacetic acid column (25 ng) were preincubated for 10 min at room temperature with 0.5 units of RNasin (Promega) in 3 mM MgCl2 in a volume of 5 l. Rubisco Purification—Wild type C. reinhardtii cells (CC-125) were grown in Tris acetate phosphate medium (3 liters), harvested to yield
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