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Characterization of a spinach chloroplast sequence‐specific DNA‐binding factor for photosystem I psa A operon promoter
Author(s) -
Wu SangPin,
Cheng MingChih,
Grace Chen ShuChen
Publication year - 1999
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1999.106114.x
Subject(s) - operon , affinity chromatography , biology , spinacia , biochemistry , chloroplast dna , dna , microbiology and biotechnology , chloroplast , binding protein , spinach , binding site , gene , escherichia coli , enzyme
The plastid genes psa A , psa B 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 mechanism of platid transcription during plant development, a DNA‐binding protein has been characterized and partially purified from spinach ( Spinacia oleracea 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 chloroplasts, ammonium sulfate fractionation (25–65% saturation), heparin agarose chromatography, heat treatment at 65°C and sequence‐specific DNA‐affinity chromatography. Gel filtration in combination with Southwestern blot analysis conclude that this DNA‐binding protein is dimeric and composed of two 34‐kDa subunits. A 28‐kDa protein without region U‐binding activity was observed to copurify with the DNA‐binding protein using DNA‐affinity chromatography.