Primary structure of barley genes encoding quinone and chlorophylla binding proteins of photosystem II

Abstract

The psbA, psbD and psbC genes encoding the quinone binding D-1 and D-2 apoproteins and the 44 kD chlorophyll a-apoprotein 3 have been located in the chloroplast genome of barley. They are found on a 23 kbp SalI restriction endonuclease fragment in the large single copy region of the chloroplast DNA adjacent to the inverted repeat. As in other species the psbD and psbC genes have reading frames which overlap by 53 bp. They are transcribed in the same direction but translated with a frameshift of one nucleotide. Ten amino acid substitutions are found among the 18 N-terminal residues of the D-2 polypeptide if barley, spinach, tobacco, pea and the liverwort Marchantia are compared. Only 8 substitutions are present among the 335 other residues of the D-2 polypeptide. The amino acid residues located in the putative binding site for the special pair reaction center chlorophyll and the residues probably serving as ligands to non-heine iron in the D-I and D-2 proteins of barley are strictly conserved when compared to those of purple bacteria and of other higher plants. Identity is also observed for the residues of importance in the binding of quinones.

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

“…The best yield was obtained with the psbD gene construct pGem 101-7, which contained only 36 bp of the untranslated 5' leader sequence and 99 bp downstream of the stop codon. The leader sequence included the Shine-Dalgarno sequence -GGAGGA-in position -11 to -6 from the initiation codon (21).…”

“…Location of the psbD gene and part of the psbC gene on a 2.23 kb EcoRI/BamHl fragment of barley cpDNA as determined by nucleotide sequence analysis (21). The position of relevant recognition sites for restriction enzymes are shown.…”

“…2 (kindly provided by PETER NIXON, Cambridge) has been raised against a truncated D-2 polypeptide, lacking the first 8 amino acids, fused to 13-galactosidase (personal communication and ref. 21). Comparison of the patterns of immunoprecipitated translation products with either of the antisera revealed no differences in antibody specificity (Fig.…”

“…Plastoquinone QA binds to two helices in the subunit M while Qa is found extended between two helices of subunit L. The primary structure of the D-1 and D-2 polypeptides deduced from the corresponding genes psbA (13,21,27) and psbD (1,9,21,26,27) in chloroplast DNA has revealed pronounced similarities of the location of certain amino acids and of the distribution of hydrophobic domains to those of the L and M polypeptides (18). It has therefore been suggested that in the reaction center complex of photosystem 2 in higher plants D-1 corresponds functionally to the L subunit and I)-2 to the M subunit of the purple bacteria (17,18,29,30) and that they form a heterodimer in the thylakoid membrane.…”

“…Towards this end I have cloned the psbD and part of the psbA gene from barley and determined their nucleotide sequence (21). In the present paper I present the construction of expression plasmids for the psbD gene and an analysis of the products obtained by in vitro transcription and translation from them.…”