Characterization and in vitro expression of the cytochromeb-559 genes of barley. II. In vitro transcription and translation

Abstract

The two cytochrome b-559 aproproteins of 9.4 kD and 4.5 kD molecular weight have been expressed in vitro using DNA templates containing either the two genes psbE and psbF in tandem or the individual genes. Transcription with E. coli RNA-polymerase or SP6 RNA-polymerase has been followed by translation in E. coli derived lysates. Simultmaeous as well as independent synthesis of the apoproteins is possible. A 9.4 kD in vitro translation product has been identified as apoprotein I by immunoprecipitation with a monoelonal antibody specific for the C-terminal part of the 9.4 kD apoprotein of cytochrome b-559. The isolated psbF gene directs the synthesis of a translation product with a molecular weight of 4.5 kD corresponding to apoprotein II. Expression of the psbE gene requires the presence of endogenous regulatory sequences 5' upstream of psbE, while this is not the case for psbF. Additional in vitro translation products of 5.7 and 2.4 kD molecular weights are synthesized and probably translated from two reading frames starting with two different out-of-phase ATG codons in the nucleotide sequence of the psbE gene.