Translational activation by a synthetic PPR protein elucidates control ofpsbAtranslation in Arabidopsis chloroplasts

Abstract

AbstractTranslation initiation onpsbAmRNA in plant chloroplasts scales with light intensity, providing its gene product, D1, to replace photodamaged D1 in Photosystem II. ThepsbAtranslational activator HCF173 has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with thepsbA5’-untranslated region (5’-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173’s footprint in thepsbA5’-UTR bound the intended sitein vivoand partially substituted for HCF173 to activatepsbAtranslation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulatepsbAtranslation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts.One sentence summaryA synthetic PPR protein substitutes for HCF173, a non-PPR translational activator in chloroplasts, elucidating HCF173 functions and demonstrating the ability of synthetic PPRs to activate translation.