Characterization of a spinach chloroplast sequence-specific DNA-binding factor for photosystem I psaA operon promoter

The plastid genes psaA, psaB and rps14 encoding the photosystem I reaction center chlorophyll proteins and ribosomal protein CS14, are organized into an operon on the circular plastid genome, To elucidate the regulatory mecha nism of platid transcription during plant development, a DNA-binding protei n has been characterized and partially purified from spinach (Spinacia oler acea L. var. spinosa Ashers et Graeden) chloroplasts, The protein interacts specifically with promoter sequences from - 51 to - 17 (named region U) of the operon. The purification procedures include high-salt extraction of ch loroplasts, ammonium sulfate fractionation (25-65% saturation), heparin aga rose chromatography, heat treatment at 65 degrees C and sequence-specific D NA-affinity chromatography. Gel filtration. in combination with Southwester n blot analysis conclude that this DNA-binding protein is dimeric and compo sed of two 34-kDa subunits, A 28-kDa protein without region U-binding activ ity was observed to copurify with the DNA-binding protein using DNA-affinit y chromatography.