Nucleotide sequence of the gene for the M _r 32,000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of M _r 38,950

Abstract

The gene for the so-called Mr 32,000 rapidly labeled photosystem II thylakoid membrane protein (here designated psbA) of spinach (Spinacia oleracea) chloroplasts is located on the chloroplast DNA in the large single-copy region immediately adjacent to one of the inverted repeat sequences. In this paper we show that the size of the mRNA for this protein is '=1.25 kilobases and that the direction of transcription is towards the inverted repeat unit. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of Mr 38,950. The nucleotide sequence of psbA from Nicotiana debneyi also has been determined, and comparison of the sequences from the two species shows them to be highly conserved (>95% homology) throughout the entire reading frame. Conservation ofthe amino acid sequence is absolute, there being no changes in a total of 353 residues. This leads us to conclude that the primary translation product of psbA must be a protein of Mr 38,950. The protein is characterized by the complete absence of lysine residues and is relatively rich in hydrophobic amino acids, which tend to be clustered. Transcription ofspinach psbA starts about 86 base pairs before the first ATG codon. Immediately upstream from this point there is a sequence typical of that found in E. coli promoters. An almost identical sequence occurs in the equivalent region of N. debneyi DNA.

Isolation of the photosystem II reaction center complex from barley. Characterization by circular dichroism spectroscopy and amino acid sequencing

“…Moreover the size of the isolated peptides was in agreement with the positions of glutamic and aspartic acid residues predicted from the gene sequence. That the barley sequence is so similar to the deduced spinach sequence and that an antibody raised against Chlamydomonas will precipitate an in vitro translated spinach protein supports our proposal in the introduction that Chl~-protein 2 is conserved to the same extent as the D2 and the 32 kD herbicide-binding polypepfide (3,13,15,22,37,50). In this connection it should be mentioned that the isolated peptides were derived from a relatively hydrophilic part of the molecule as judged from the deduced spinach protein sequence.…”

“…The 33 kD polypeptide stained well with Coomassie Brilliant Blue. When its amino acid composition was determined (Table III), it was found to contain lysine, indicating that this 33 kD polypeptide is not related to the quinonebinding polypeptide of similar size since the latter is completely deficient in lysine (50). The amino acid compositions of the 33 kD polypeptide associated with the PSII reaction center complex and of the 32 kD polypeptide from the oxygen-evolving system described in (30) are similar, indicating that they are one and the same polypeptide.…”

“…The nucleotide sequence of this gene has been determined in a variety of organisms, showing very few differences between cyanobacteria (13), Chlamydomonas (15) and higher plants (50). The sequence of the D2 membrane polypeptide from PSII is also conserved (3,22,37).…”

A thylakoid polypeptide involved in the reconstitution of photosynthetic oxygen evolution

“…An M r 32,000 polypeptide has also been implicated on the reducing side ofphotosystem II and identified as the protein which binds DCMU and atrazin (34,39). The nucleotide sequence of the gene coding for this M r 32,000 polypeptide is known for spinach and tobacco and an absolute conservation of the amino sequence was found (52). The protein contains no lysine and is thus clearly different from the M r 32,000 polypeptide isolated in this study (Table III).…”

“…The protein contains no lysine and is thus clearly different from the M r 32,000 polypeptide isolated in this study (Table III). Further the DCMU-binding protein does not appear to stain with Coomassie Blue (33,52).…”