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

Abstract

The Escherichia coli RNA polymerase (RNAP) α, β, and β′ 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 α subunit gene (rpoA) with the E. coliα subunit gene by targeted gene insertion. The transplastomic tobacco plants look similar to tobacco rpoA deletion mutants in that they are chlorophyll-deficient and non-photoautotrophic. 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 α subunits is insufficient to allow substitution of the tobacco α subunit with its bacterial counterpart. Interestingly, the cyanobacterial α subunits are as different as the E. coliα subunits; and therefore it is unlikely that replacement of the tobacco α subunit with cyanobacterial α subunits would yield a functional enzyme. Replacement of plastid rpoA with the E. coli RNA polymerase α subunit gene represents the first engineering of a plastid operon in higher plants.