Using bacteria to analyze sequences involved in chloroplast gene expression

Abstract

The expression of higher plant chloroplast genes in prokaryotic cells has been used to examine organelle sequences involved in promoter recognition by RNA polymerase, and protein translocation through membranes. The similarity in sequence structure between Escherichia coli promoters and the maize chloroplast atpB promoter has been investigated using deletion and single base pair substitution mutants. The atpB mutants were mainly isolated by a selection system in E. coli, and then used as templates for the analysis of transcription using chloroplast RNA polymerase. It was found that both the bacterial and chloroplast RNA polymerases behaved in a similar fashion with the wild-type and mutant promoters, indicating that the sequences involved in promoter recognition share a considerable degree of homology. Signal peptide recognition of pea cytochrome f has also been examined in E. coli. This signal peptide, which is probably responsible for insertion of the protein into the thylakoid membrane, is efficiently recognized in E. coli leading to the inner membrane insertion of petA::lacZ fusion proteins. This process requires the bacterial SecA protein and points to a general similarity in the mechanisms of protein translocation within chloroplasts and bacteria.