Nucleic Acids Research, 1984, Vol. 12, No. 15 6221-6230
© 1984
MOLECULAR BIOLOGY |
In vitro expression and characterization of the translation start site of the psbA gene product (QB protein) from higher plants
Roche Institute of Molecular Biology, Roche Research Center Nutley, NJ 07110, USA
Received February 9, 1984. Revised July 9, 1984. Accepted July 9, 1984.
The psbA gene from higher plants, which codes for the atrazine herbi cide 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.