In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley

Abstract

The plastoquinone-binding polypeptide D-2 of the reaction center complex of photosystem II in barley has been expressed in vitro using the expression vector pGem3 TM containing the psbD gene of chloroplast DNA as template. The full length D-2 polypeptide with an apparent mol. weight of 32 kD is a major product when mRNA is transcribed with SP6 RNA polymerase from an insert containing the psbD gene with a 36 bp 5' leader sequence and a 3' tail of 99 bp downstream of the stop codon. Translation of the mRNA had to be done in a rabbit reticulocyte lysate. Translation also occurred from the internal AUG codons giving rise to truncated D-2 polypeptides with sizes of 30, 25 and 17 kD. They are immunoprecipitable with antisera either raised against amino acid residues 235 to 241 or against 345 residues of the D-2 polypeptide. A truncated translation product of 12 kD probably initiated at codon 247 is not recognized by either antiserum. An additional immunoprecipitable protein with an apparent molecular weight of 46 kD is observed by SDS-PAGE and is interpreted as a homomeric aggregate of D-2 polypeptides. Upon addition of methanol solubilized 2,6 dichloro-p-benzoquinone or other quinones a preferential translation of the full-length and truncated D-2 polypeptides is observed. The use of in vitro synthesized D-2 polypeptides for studies of bintiing quinones, other electron carriers and chlorophyll chromophores is discussed.