Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region.
We have previously shown the presence in chloroplasts of sequence-specific DNA-binding proteins that interact specifically with two regions located downstream and upstream from the 5'-transcription start site of the plastid psaA-psaB-rps14 operon. As part of an effort to elucidate the regulatory mechanism of plastid transcription during plant development, we report here the purification and characterization of the chloroplast DNA-binding protein from spinach (Spinacia oleracea L. var. spinosa Ashers et Graeden) leaves that specifically recognizes sequences between positions +64 to +83 relative to the transcription start site. This DNA-binding protein has been highly purified from chloroplasts by using a combination of high-salt extraction, ammonium sulfate precipitation, heparin-agarose chromatography, and sequence-specific DNA-affinity chromatography. The protein exhibited an apparent molecular weight of 59-60 kDa on the basis of gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Southwestern blot analysis further indicated that this DNA-binding protein is dimeric and composed of two approximately 31-kDa subunits. We discuss the properties of this protein in relation to the known chloroplast DNA-binding factors for plastid gene expression.
Cheng
M C
MC
Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China.
Wu
S P
SP
Chen
L F
LF
Chen
S C
SC
D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D012275:Oryza / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis / Q000235:genetics*; D045331:Photosystem I Protein Complex; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D018087:Plastids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D018724:Spinacia oleracea / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D010940:Plant Proteins; C089909:photosystem I, psaB subunit