Identification and purification of a spinach chloroplast DNA-binding protein that interacts specifically with the plastid psaA-psaB-rps14 promoter region

Abstract.

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 ≈31-kDa subunits. We discuss the properties of this protein in relation to the known chloroplast DNA-binding factors for plastid gene expression.