Engineering of the rpl23 gene cluster to replace the plastid RNA polymerase alpha subunit with the Escherichia coli homologue.

Abstract

The Escherichia coli RNA polymerase (RNAP) alpha, beta, and beta' core subunits are evolutionarily conserved among bacteria and plastids, and the plastid specificity factors form a functional holoenzyme with the E. coli core. To investigate whether the E. coli core subunits may form a functional hybrid enzyme with the plastid core subunits, we replaced the tobacco plastid RNAP alpha subunit gene (rpoA) with the E. coli alpha subunit gene by targeted gene insertion. The transplastomic tobacco plants look similar to tobacco rpoA deletion mutants in that they are chlorophyll-deficient and nonphotoautotrophic. In addition, they lack transcripts from promoters recognized by the E. coli-like plastid RNA polymerase. These results indicate that evolutionary conservation between the E. coli and plastid RNA polymerase alpha subunits is insufficient to allow substitution of the tobacco alpha subunit with its bacterial counterpart. Interestingly, the cyanobacterial alpha subunits are as different as the E. coli alpha subunits; and therefore it is unlikely that replacement of the tobacco alpha subunit with cyanobacterial alpha subunits would yield a functional enzyme. Replacement of plastid rpoA with the E. coli RNA polymerase alpha subunit gene represents the first engineering of a plastid operon in higher plants.