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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.

The 32 kDa herbicide binding protein is a membrane bound protein which is implicated in the binding of many photosystem II herbicides as well as in the binding of the endogenous quinone OB which serves as the secondary electron acceptor on the reducing side of photosystem II. The topology of the 32 kDa protein has been predicted using a combination of hydrophobic moment analysis, membrane propensity analysis and empirical secondary structure predictions. Our model consists of five transmembrane helices. The loop connecting the fourth and fifth transmembrane helices is thought to form part of the herbicide binding site. Our analysis suggests that this loop also contains a helical segment which may seek the surface of the membrane by virtue of its relatively high hydrophobic moment. Our topology is compared with several others which have been proposed in the literature as well as with the topology of the L and M proteins of the bacterial reaction center of R. viridis. The significance of mutagenesis and photo-affinity labeling experiments is also discussed in terms of our model.