RNA Binding Activity of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii

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 RNAbinding 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.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. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1-3). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4, 5).Ribulose-1,5-bisphosphate carboxylase (Rubisco) is the key enzyme in photosynthetic carbon assimilation. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family. Assembly of the holoenzyme is mediated by the chloroplast chaperonins cpn60 and cpn10 (6, 7). We previously showed that transfer of the green algae C. reinhardtii from low light (70 mol m Ϫ2 s Ϫ1 ) to high light (700 mol m Ϫ2 s Ϫ1 ) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8). These light-induced effects were found to be transient, with cell recovery taking pla...

A Conserved Mechanism Controls Translation of Rubisco Large Subunit in Different Photosynthetic Organisms

“…These values are consistent with our previous report on RNA binding by Rubisco LSU and by the recombinant N terminus of C. reinhardtii (Yosef et al, 2004).…”

“…Thus, despite the significant differences in the amino acid sequences of the Rubisco N-terminal domains, the structural alignment revealed a remarkable conservation throughout evolution. The typical basic pattern of the babbab structure was found in all the N termini of the structures, and as previously reported (Yosef et al, 2004) it is typical of the RNA recognition motif (Nagai, 1996;Siomi and Dreyfuss, 1997). The high degree of evolutionary conservation observed in the structure of the N terminus suggests that this region could play a similar role in different photosynthetic organisms, possibly in the complex regulation of the assembly process and subunit expression.…”

“…While searching for regulatory components that interact with the rbcL transcript we found that under oxidizing conditions, the Rubisco LSU can bind RNA in vitro in a sequence-independent manner and that the N terminus (150 amino acids) is responsible for this activity. Under normal conditions, the N terminus is buried in the folded protein, but upon oxidation of the protein it becomes exposed and will bind any RNA in the vicinity (Yosef et al, 2004). Indeed, under oxidizing conditions, the rbcL transcript remains in association with ribosomes but shifts toward the monosomal fraction.…”

“…A similar labeling pattern was obtained for spinach (Spinacia oleracea) leaf discs (data not shown). We recently proposed that exposure of the N-terminal domain (1-150 amino acids) of Rubisco LSU from C. reinhardtii to oxidizing conditions, and the consequent binding of RNA in a sequence-independent manner (Yosef et al, 2004), plays a role in the regulation of Rubisco expression. The N terminus of Rubisco LSU is not involved in CO 2 fixation, as the active site of the enzyme lies in the C-terminal domain, which has the structure of an eight-stranded a/b barrel (TIM barrel).…”

Constraining the timing of the Great Oxidation Event within the Rubisco phylogenetic tree

“…It comprises an RNA recognition motif that becomes exposed under oxidizing conditions when the glutathione pool shifts toward its oxidized form (Cohen, Sapir, & Shapira, 2006). The oxygen sensitivity of the N‐terminus, and its role binding to RNA, may play an evolutionarily significant role in regulating Rubisco expression (Cohen et al., 2006; Kapralov & Filatov, 2007; Yosef et al., 2004). Regulation of expression can attenuate acute physiological response to perturbation inside or outside the cell (Bailey, 1991; Berry, Mure, & Yerramsetty, 2016).…”