RNA Binding Activity of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit from Chlamydomonas reinhardtii
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m308602200
ISSN1083-351X
AutoresIdo Yosef, Vered Irihimovitch, Joel A. Knopf, Idan Cohen, Irit Orr-Dahan, Eyal Nahum, Chen Keasar, Michal Shapira,
Tópico(s)Photoreceptor and optogenetics research
ResumoTransfer 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. 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. When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RB, RNA binding; EMSA, electrophoretic mobility shift assays; SK, pBluescript; RBD, RNA binding domain; LSU, large subunit; UTR, untranslated region; DTT, dithiothreitol; aa, amino acids. that cause a variety of cellular and molecular damage. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1Barber J. Andersson B. Trends Biochem. Sci. 1992; 17: 61-66Abstract Full Text PDF PubMed Scopus (843) Google Scholar, 2Osmond C.B. Baker N.R. Boyer J.R. Photoinhibition of Photosynthesis, from Molecular Mechanisms to the Field. BIOS Scientific Publishers Ltd., Oxford1994: 1-24Google Scholar, 3Prásil O. Adir N. Ohad I. Barber J. The Photosystems: Structure, Function, and Molecular Biology. Elsevier Science Publishers B. V., Amsterdam1992: 295-348Crossref Google Scholar). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4Foyer C.H. Lopez-Delgado H. Dat J.F. Scott I.M. Physiol. Plant. 1997; 100: 241-254Crossref Google Scholar, 5Noctor G. Gomez L. Vanacker H. Foyer C.H. J. Exp. Bot. 2002; 53: 1283-1304Crossref PubMed Scopus (689) Google Scholar). 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 (6Gutteridge S. Gatenby A.A. Plant Cell. 1995; 7: 809-819Crossref PubMed Scopus (139) Google Scholar, 7Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (329) Google Scholar). We previously showed that transfer of the green algae C. reinhardtii from low light (70 μmol m–2 s–1) to high light (700 μmol m–2 s–1) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8Shapira M. Lers A. Heifetz P. Yrihimovitz V. Osmond B.C. Gillham N.W. Boynton J.E. Plant Mol. Biol. 1997; 33: 1001-1011Crossref PubMed Scopus (70) Google Scholar). These light-induced effects were found to be transient, with cell recovery taking place within 6–12 h once chlorophyll levels were reduced and ROS levels were decreased. It was further found that translation of Rubisco LSU varies with the changes in ROS production and correlates with alterations in the ratio between oxidized and reduced glutathione. Upon transfer to high light the glutathione pool shifts to its oxidized form, and LSU synthesis stops almost completely. When the cells recover from light stress, the glutathione pool shifts back to its reduced form, and LSU translation resumes (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Rubisco holoenzyme is highly susceptible to oxidative stress in vivo. Excess ROS caused a rapid translocation of the soluble enzyme complex into the chloroplast membrane and the formation of intermolecular cross-linking between the large subunits via disulfide bonds (10Mehta R.A. Fawcett T.W. Porath D. Mattoo A.K. J. Biol. Chem. 1992; 267: 2810-2816Abstract Full Text PDF PubMed Google Scholar). Furthermore, oxidative stress can cause direct fragmentation of Rubisco LSU at Gly-329 into 37- and 16-kDa polypeptides in illuminated intact chloroplasts in chloroplast extracts and in its purified form when exposed to a hydroxyl radical-generating system (11Ishida H. Makino A. Mae T. J. Biol. Chem. 1999; 274: 5222-5226Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Translational regulation allows plants to respond quickly to environmental changes such as light intensities and is, therefore, a predominant mechanism in chloroplasts. Upstream UTRs are expected to play a key role in translation of chloroplast genes via interaction with regulatory proteins. A group of RNA binding (RB) proteins with molecular masses of 60, 55, 47, and 38 kDa assemble on the 5′-UTR of the psbA RNA (12Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4002Crossref PubMed Scopus (165) Google Scholar). RB47 shows a high homology with the eukaryotic poly(A)-binding protein (13Yohn C.B. Cohen A. Danon A. Mayfield S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2238-2243Crossref PubMed Scopus (86) Google Scholar), and RB60 shows high homology to protein disulfide isomerase (14Kim J. Mayfield S.P. Science. 1997; 278: 1954-1957Crossref PubMed Scopus (198) Google Scholar, 15Trebitsh T. Meiri E. Ostersetzer O. Adam Z. Danon A. J. Biol. Chem. 2001; 276: 4564-4569Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Binding of the protein complex is mediated by specific thiol-containing proteins, and light-induced reduction of thioredoxin enhances this binding. It was proposed that RB60 serves as a redox sensor that activates binding of RB47 (16Trebitsh T. Levitan A. Sofer A. Danon A. Mol. Cell. Biol. 2000; 20: 1116-1123Crossref PubMed Scopus (105) Google Scholar). Proteins with similar molecular masses assemble on the 5′-UTR of psbC and other chloroplast leaders (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Zerges W. Rochaix J.D. J. Cell Biol. 1998; 140: 101-110Crossref PubMed Scopus (100) Google Scholar), although it is possible that different RNA-binding proteins of size 47 kDa have altered target specificities (19Zerges W. Wang S. Rochaix J.D. Plant Mol. Biol. 2002; 50: 573-585Crossref PubMed Scopus (12) Google Scholar). In an attempt to examine the mechanism that underlies the unique pattern of regulation observed for Rubisco LSU, we examined how the redox state of RNA-binding proteins affected their interaction with the rbcL leader. We show that the interaction between RNA-binding proteins and the rbcL leader is interrupted by oxidative stress and that Rubisco LSU can bind RNA in its oxidized form, although in a nonspecific manner. 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. Cultures were illuminated with medium light (150 μmol m–2 s–1) using cool white fluorescent lamps. The photosynthesis-deficient mutant CC-2653 has an amber mutation at the 5′ end that terminates translation of Rubisco LSU at position 65 (20Spreitzer R. Goldschnidt-Clermont M. Rahire M. Rochaix J.-D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Crossref PubMed Google Scholar) and was grown on Tris acetate phosphate medium at 25 °C under dark conditions. For Rubisco purification CC-125 cells were grown in Tris acetate phosphate medium under low light conditions (70 μmol m–2 s–1). Plasmids Used for in Vitro RNA Synthesis—The 5′-UTR of rbcL from C. reinhardtii was cloned from P-266, a plasmid that contains the 4-kilobase EcoRI-BamHI fragment of chloroplast DNA (21Dron M. Rahire M. Rochaix J.D. J. Mol. Biol. 1982; 162: 775-793Crossref PubMed Scopus (179) Google Scholar). An EcoRI-XmnI fragment of the insert was inserted into pBluescript at the EcoRI-SmaI site, resulting in pVI1. The complete 5′-UTR of rbcL (between positions –93 to +24 relative to the ATG start codon) was amplified from pVI1 using the primers 5′-TAAATGTATTTAAAATTTTTCAACAAT-3′ (forward) and 5′-TTTAGTTTCTGTTTGTGGAACCAT-3′ (reverse). The resulting PCR fragment was cloned into the pGEM-T vector (Promega), and sequences between the PstI and NsiI sites were removed, resulting in pΔVI3. The plasmid encoding rbcL was linearized by NcoI and NotI for synthesis of the sense and antisense strands, respectively. In vitro synthesis of the 5′-UTRs derived from psbA, atpB (chloroplast genes), and α-tubulin (a nuclear gene) was performed from plasmids D1-HA, P-419, and P-654, respectively. Plasmid D1-HA was the generous gift of A. Danon; plasmids P-419 and P-654 were obtained from the Chlamydomonas Center at Duke University. Plasmid D1-HA was linearized with EcoRI, and P-419 and P-654 were linearized with BamHI. In vitro synthesis of the sense strands derived from the corresponding 5′-UTRs was performed with T7 RNA polymerase. Synthesis of a non-related RNA fragment was performed using pBluescript (SK) as a template. The plasmid was linearized by NotI, and RNA was synthesized with T7 RNA polymerase. Preparation of Protein Extracts—C. reinhardtii wild type CC-125 cells were grown at medium light, and protein extracts were prepared essentially as described previously (12Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4002Crossref PubMed Scopus (165) Google Scholar, 17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Cells (3 × 1 liter) were grown in high salt reduced sulfate and harvested at a concentration of 5–7 × 106 cell/ml. The pellets were frozen in liquid nitrogen and stored at –70 °C until use. Cell pellets (8 g fresh weight) were thawed in 25 ml of low salt buffer (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl2, and 10 mm DTT) in the presence of protease inhibitors (2 μg/ml aprotinin, 10 μg/ml benzamidine, 5 μg/ml leupeptin, and 76 μg/ml phenylmethylsulfonyl fluoride). The cells were disrupted by triple passage through a French press (Sim-Amico, Spectronic Instruments) at 4000 p.s.i. The broken cells were centrifuged for 10 min at 20,000 × g (SS-34 rotor, 10,000 rpm in an RC-2 Sorvall). The supernatants were collected and further centrifuged at 200,000 × g for 1 h at 4 °C (TI50 rotor, 50,000 rpm in an L8–55 Beckman ultracentrifuge). The 200,000 × g supernatant was collected and applied immediately onto a 5-ml heparin-Actigel column (Amersham Biosciences) at a flow rate of 1 ml/min. The column was first prewashed with 3 volumes of high salt buffer (10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl2, 10 mm DTT, and 2 m potassium acetate) and equilibrated with extraction buffer containing (20 mm Tris-HCl, pH 7.5, 3 mm MgCl2, 0.1 mm EDTA, and 2 mm DTT). The bound proteins were eluted with a continuous gradient of potassium acetate concentrations (0–1.6 m) in low salt buffer. Fractions (500 μl) were collected, dialyzed against dialysis buffer (20 mm Tris-HCl, pH 7.5, 100 mm potassium acetate, 0.2 mm EDTA, 2 mm DTT, and 20% glycerol), and stored at –70 °C. The fractions were analyzed by SDS-PAGE (12%), and their protein content was evaluated by Coomassie Blue staining. In Vitro RNA Synthesis—Radiolabeled RNA transcripts (described above) were synthesized in vitro using 0.5–1 μg of DNA in 40 mm Tris-HCl, pH 7.5, 6 mm MgCl2, 2 mm spermidine, 12.5 mm NaCl, 10 mm DTT, 20 units of RNasin, 0.5 mm ATP, GTP, and CTP, 12 μm UTP, 50 μCi of [α-32P]UTP (800 Ci/mmol, Amersham Biosciences), and 20 units of SP6 (Roche Applied Science) or T7 (Promega) RNA polymerase in a reaction volume of 20 μl. The reactions were performed at 37 °C for 40 min followed by the addition of 1 unit of DNase I (RNase-free, Promega) and were then incubated for an additional 30 min at 37 °C. The labeled RNAs were separated from the unincorporated ribonucleotides on a spun-down mini-column of Sephadex G-50 in double-distilled H20. Under these conditions transcripts were labeled to specific activities that ranged between 5 × 108 and 2 × 109 cpm/μg of RNA. Unlabeled transcripts that were used for competition assays were synthesized as described above, except that the reactions were scaled up to 100 μl, and all the four ribonucleotides were included at equal concentrations (0.5 mm). RNA products were analyzed on 7 m urea, 6% polyacrylamide gels to verify production of a single transcript and to evaluate its size and concentration. Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSA) were performed essentially as described previously (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Samples from the heparin-Actigel column fractions (2 μl containing approximately 9 μg 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 μl. The mixtures were then added to the RNA probes (10,000 cpm) in the presence of 20 μg of Escherichia coli tRNA (Sigma) in a final volume of 15 μl. After an incubation of 15 min at room temperature, 2 μl of loading buffer (0.25 μg/μl xylene cyanol, 0.25 μg/μl bromphenol blue, and 6% (v/v) glycerol) were added, and the reactions were separated on a native 5% polyacrylamide gel (acrylamide:bisacrylamide 49:1) in 1× TBE (89 mm Tris, 89 mm boric acid, and 2 mm EDTA, pH 8). Running conditions were 25 mA for 2–3 h. The gels were then fixed in a solution of 20% methanol and 10% acetic acid, dried, and subjected to autoradiography. In competition experiments unlabeled RNA transcripts were added in varying amounts of mass excess and preincubated with the protein samples before the addition of the radio-labeled probe. UV Cross-linking Assays—Binding assays were performed as previously described (17Hauser C.R. Gillham N.W. Boynton J.E. J. Biol. Chem. 1996; 271: 1486-1497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Samples of proteins eluted from the heparin-Actigel column (2 μl containing approximately 9 μg 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 μl. Radiolabeled RNA (100,000 cpm) was then added to the protein solution along with 0.2 μg of E. coli tRNA (Sigma) in a final volume of 15 μl. After 15 min of incubation at room temperature the binding reactions were placed on ice and cross-linked by UV irradiation at 254 nm in a UV cross-linker (Hoefer) for 90 s. RNA transcripts were then digested with 20 μg of RNase A (Sigma) for 40 min at 37 °C. The samples were separated over 15% SDS-PAGE. Gels were stained by Coomassie Blue, dried, and subjected to autoradiography or analyzed by phosphorimaging. In competition experiments unlabeled RNA transcripts were added in varying mass excess and preincubated with the protein samples before the addition of the radiolabeled probe. To examine how the redox state affected the binding proteins, GSSG or GSH was added to the protein extracts or to the purified Rubisco (25 ng) 10 min before the addition of the labeled 5′-UTR. Two-dimensional Gel Electrophoresis—Protein separation on two-dimensional gel electrophoresis was performed as previously described (22O'Farrel P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar). Separation on the first dimension was performed by isoelectric focusing using ampholytes (Bio-Rad) that ranged between pH 3 and 9. Separation on the second dimension was preformed by SDS-PAGE over 12% polyacrylamide gels. Antisera—Polyclonal rabbit antisera raised against Rubisco holoenzyme from tobacco were a generous gift of T. J. Andrews from the Australian National University, Canberra, Australia. Western Blot Analysis—Proteins were separated over one- and two-dimensional SDS-polyacrylamide gels and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell). Western blot analysis was performed using anti-Rubisco antibodies (1:4000) and a conjugate of protein A with alkaline phosphatase (1:2000). Antibody binding was detected with nitro blue tetrazolium and 5′-bromo-4-chloro-3-indolylphosphate (Sigma) dissolved in a buffer containing 100 mm Tris-HCl, pH 9.5, 100 mm NaCl, and 5 mm MgCl2. Rubisco Purification—Wild type C. reinhardtii cells (CC-125) were grown in Tris acetate phosphate medium (3 liters), harvested to yield 8 g fresh weight, and disrupted in a French press as describe above. The cell extract was loaded on a heparin-Actigel column pre-equilibrated in low salt buffer (see above) and was eluted by a single step of 1.6 m potassium acetate in low salt buffer. The eluate (5 ml) was dialyzed against low salt buffer and loaded (1 ml) on a linear 10–30% sucrose gradient in low salt buffer. The gradient was centrifuged for 16 h at 164,000 × g (SW40 rotor, 40,000 RPM in a Beckman LE-80K ultracentrifuge) at 4 °C, and fractions (1 ml) were collected. Samples of each fraction were examined by Western blot analysis using antibodies raised against Rubisco. Expression of Recombinant LSU and Its Subdomains in Bacteria— The DNA region that encodes for Rubisco LSU was amplified using primers derived from both ends of the rbcL gene; the LSU-fwd-(1–21) primer was 5′-ATCCATGGTTCCACAAACAGAAACT-3′, and the LSU-rev-(1506–1482) primer was 5′-TTAGGAATTCAACGTAAACACCATA-3′. The DNA region which encodes the amino terminus of Rubisco LSU (aa 1–150) was amplified by PCR using the LSU-fwd-(1–21) primer; and the reverse primer, LSU-rev-(450–432), 5′-GGAATTCTTAACCTACGAATGTTTTAACG-3′. The DNA region that encodes the TIM barrel domain of Rubisco (aa 151–475) was amplified by PCR using the primer LSU-fwd-(451–466), 5′-ATCCATGGAACCTCCACACGGTATTC-3′, and the LSU-rev-(1506–1482) primer. Anchors that introduced restriction sites, NcoI (fwd primers) and EcoRI (rev primers), were added and are marked in bold letters. The resulting fragments were cloned in-frame into the parallel pHisII expression vector (23Sheffield P. Garard S. Derewenda Z. Protein Expression Purif. 1999; 15: 34-39Crossref PubMed Scopus (530) Google Scholar). Expression was induced in BL21 cells by incubation of the transgenic bacteria with 0.3 mm isopropyl-1-thio-β-d-galactopyranoside at 20 °C for 2.5 h. The bacteria were harvested, washed once in double-distilled H2O, resuspended in a buffer containing 30 mm Tris-HCl, 300 mm NaCl, and 5 mm imidazole, and lysed in a French press using 20,000 p.s.i. The lysate was centrifuged at 10,000 × g at 4 °C for 25 min. The expressed protein present in the soluble bacterial fraction was affinity-purified over a nickel nitrilotriacetic acid column (Qiagen). Because the recombinant TIM barrel domain was insoluble, it was denatured after a previously described procedure (24Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual.3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 15.49-15.54Google Scholar) and renatured by dialysis against 50 mm Tris-HCl, pH 8.5, 10 mm MgCl2,10 mm NaHCO3, 1 mm DTT. The soluble recombinant polypeptides were subjected to Western blot analysis using antibodies against Rubisco. Structural Characterization of Rubisco Subdomains—Subdomains of the large subunit of Rubisco were identified based on the SCOP data base that provides a structural classification of proteins (25Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5595) Google Scholar). RNA Binding Activity of Protein Extracts to the rbcL 5′-UTR; Specificity and Sensitivity to the Redox State—To test whether translational arrest of the rbcL transcript at high light and during oxidative stress correlated with changes in the redox state of proteins that bind to its 5′-UTR, electrophoretic mobility shift assays were performed. The fraction of proteins that bind nucleic acids was enriched by purification of cell extracts over a heparin-Actigel affinity column followed by elution with a gradient (0–1.6 m) of potassium acetate concentrations. Radiolabeled RNA extending from positions –93 to +24 relative to the translational start site of the rbcL gene was synthesized in vitro and incubated with the protein fractions. Binding of proteins to the radiolabeled RNA was monitored by their inhibitory effect on migration of the RNA in native polyacrylamide gels as compared with control unbound RNA. RNA binding activity was observed with the radiolabeled 5′-UTR of rbcL (Fig. 1A). Migration of the RNA-protein complexes resulted in multiple bands, suggesting that the binding involved more than a single protein. Binding to the labeled 5′-UTR of rbcL was interrupted in a dose-response manner by adding increasing amounts of the corresponding unlabeled rbcL 5′-UTR (Fig. 1A, lanes b–f). Binding was not affected by the addition of increasing amounts of SK RNA (lanes h–l) or by a large excess of E. coli tRNA (lanes m and n). Thus, only the homologous non-radioactive RNA fragment could efficiently compete-out the RNA-protein interaction. In addition, no binding was observed with a nonspecific labeled RNA fragment of comparable size, derived from pBluescript (SK RNA, data not shown). To strengthen the association between the shift of glutathione to its oxidized form during light stress and the translational arrest of Rubisco LSU (9Irihimovitch V. Shapira M. J. Biol. Chem. 2000; 275: 16289-16295Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), binding assays were carried out under oxidizing (GSSG) and reducing (GSH, DTT) conditions. Fractionated proteins capable of binding to the 5′-UTR of rbcL in EMSA were preincubated with increasing concentrations of GSSG, GSH, and DTT (5, 10, and 25 mm) before the addition of the radiolabeled rbcL 5′-UTR. Band shifting of the RNA was inhibited already in the presence of 5 mm GSSG and was completely abolished by 10 and 25 mm GSSG, whereas GSH and DTT had no effect on the binding activity (Fig. 1B). These results indicate that mediating the redox of thiol groups on RNA-binding protein(s) affects their ability to interact with the 5′-UTR of the rbcL transcript. Binding Specificity of Proteins to the 5′-UTR of rbcL—A biochemical characterization of the proteins that bind to the rbcL leader was initiated using UV cross-linking. Radiolabeled RNA corresponding to the 5′-UTR of rbcL was incubated with proteins eluted from the heparin column and subjected to UV irradiation followed by RNase digestion. Using this approach we found that proteins with a molecular mass of 81, 62, 51, 47, 38, 36, and 34 kDa bind to the 5′-UTR of rbcL. To establish that the binding was specific to the 5′-UTR of rbcL, UV cross-linking was performed in the presence of non-radioactive RNA competitors. Binding of the 81-, 62-, 51-, and 47-kDa proteins was inhibited by the homologous rbcL –93/+24 fragment (Fig. 2A, lanes a–g) and was hardly affected by the SK RNA or tRNA controls (Fig. 2A, lanes h–q), indicating a sequence specificity for the RNA-protein interactions. Because binding of the 38-, 34-, and 32-kDa proteins was not competed-out by the cold rbcL RNA, their interaction with the RNA was most probably nonspecific. UV Cross-linking of Proteins to the 5′-UTR of rbcL Is Sensitive to the Redox State of Glutathione—To test how the redox state of thiol groups modulated binding of proteins to the 5′-UTR of rbcL, UV-cross-linking assays were performed in the presence of reduced and oxidized glutathione. Protein extracts eluted from the heparin-Actigel column were preincubated with increasing concentrations of GSSG before the addition of the labeled rbcL 5′-UTR. Under oxidizing conditions binding of the 81-, 62-, 51-, and 47-kDa proteins decreased (Fig. 2B); however, cross-linking of a new 55-kDa protein was observed. In view of the similar molecular weights of this newly UV-cross-linked protein and the LSU of Rubisco, the possibility that an autoregulatory pathway exists was considered. Rubisco LSU Is an RNA-binding Protein—The 55-Da protein was characterized on one- and two-dimensional polyacrylamide gels combined with Western blot analysis. Proteins were preincubated with GSSG (7.5 mm) before their UV cross-linking with the radiolabeled 5′-UTR of rbcL. The cross-linked proteins were separated by two-dimensional SDS-PAGE and blotted onto nitrocellulose membranes. The blots were exposed to a film (Fig. 3, C and D) and then reacted with an antibody raised against Rubisco LSU (Fig. 3, A and B). Alignment of the autoradiograms and films indicated that the 55-kDa protein that cross-linked to the 5′-UTR of rbcL under oxidizing conditions co-migrated with Rubisco LSU on two-dimensional gels. Direct evidence for binding of Rubisco LSU to the rbcL leader under oxidizing conditions was obtained by using the purified enzyme in UV-cross-linking experiments. As shown in Fig. 4, A and B, the purified protein cross-linked with the rbcL leader in direct correlation with the GSSG concentration, whereas no binding was observed in the absence of GSSG. In addition, the 55-kDa protein was absent in UV-cross-linking experiments performed under oxidizing conditions using CC 2653 (20Spreitzer R. Goldschnidt-Clermont M. Rahire M. Rochaix J.-D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5460-5464Crossref PubMed Google Scholar), a mutant that fails to express Rubisco LSU due to a point mutation that pre-terminates translation (data not shown). To examine the binding specificity between Rubisco LSU and its corresponding rbcL leader, competition assays were performed with unlabeled RNAs that corresponded to the homologous rbcL leader in its sense and antisense orientations. Equal inhibition of binding to the labeled rbcL leader was observed with similar amounts of either fragment, suggesting that in vitro the binding of Rubisco LSU to its leader was sequence-independent (Fig. 5A). A similar conclusion was drawn from experiments that compared the competition between non-labeled SK RNA and the sense rbcL fragment. Both of these RNA fragments competed out the binding between Rubisco LSU and its RNA leader with comparable efficiencies (Fig. 5B). It, therefore, appears that under oxidizing conditions Rubisco LSU binds non-specifically to RNA. In the presence of a large excess of cold competitor RNA, a higher band was observed. Its appearance could result from incomplete RNase digesti
Referência(s)