In vitroexpression and characterization of the translation start site of thepsbA gene product (Q_Bprotein) from higher plants

Abstract

The psbA gene from higher plants, which codes for the atrazine herbicide binding protein of photosystem II (QB protein), has been recently sequenced by various laboratories. From these data there are two potential translation sites, one yielding a protein of 38,500 kd and another a protein of 34,500 kd. In the present study, cloned psbA gene sequences from maize, tobacco, and pea have been expressed in a highly defined E. coli in vitro transcription/translation system. In order to determine the start site of translation, we also have employed a simplified E. coli system designed to synthesize the first di- or tripeptide of the gene product. From these results, it is clear that the first ATG of the longest open reading frame of the psbA gene, that begins fMet-Thr, is not recognized in vitro. Instead, the next downstream Met at position 37 is the initiation site, since the expected dipeptide fMet-Ile is synthesized from all psbA clones. These data are in accord with the in vivo results that the gene product is a precursor protein of 34,500 kd.

Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1

“…Moreover, the psbA gene contains a sequence similar to a prokaryotic Shine-Dalgarno sequence just upstream of the Met-37 codon. Our results correlate with data obtained earlier on the expression of psbA genes of other higher plants in prokariotic in vitro translation systems [3,14]. At the same time, in our experiments on in vitro expression of the barley psbA gene in the rabbit reticulocyte lysate system, a major expression product of A& -33.5 kDa, and only a small amount of 29 kDa protein was observed [6].…”

The chloroplast 32 kDa protein is synthesized on thylakoid‐bound ribosomes in Chlamydomonas reinhardtii

“…Cohen et al [25] have recently shown that the translation start site for this polypeptide from higher plants is an internal methionine, at position 37. The initiating methionine is followed by a highly hydrophobic stretch of about 21 amino acids at the amino terminus [21].…”

Photosynthesis and the Application of Molecular Genetics

“…The nucleotide sequence of genes isolated from a wide variety of oxygen-evolving photosynthetic organisms, including algae and cyanobacteria, has shown a remarkable conservation of amino-acid composition and order, predicting 352-360 residues (353 in higher plants) and a molecular weight of about 39 kD (Zurawski et al, 1982;Marder, 1985). There are arguments, however, that translation actually starts at 37 triplets downstream, giving a translation product of 317 residues and molecular weight 34-6 kD (Hirschberg and McIntosh, 1983;Cohen et al, 1984). A precursor with an electrophoretic mobility corresponding to 33-5 kD has been observed and is processed to the mature size by removal of about 15 residues from the carboxyl terminus (Marder, Goloubinoff and Edelman, 1984).…”