Specific DNA-binding sites of bacterial-type chloroplast RNA polymerase in young Arabidopsis seedlings

Photosynthesis-associated genes encoded in the plastid genome are mainly transcribed by plastid-encoded plastid RNA polymerase (PEP) in chloroplasts. PEP is a prokaryotic-type RNA polymerase and its DNA binding depends on sigma factors. In angiosperms, PEP also requires PEP-associated proteins (PAPs) for its function in vivo. Angiosperms possess evolutionarily distinct plastid-targeted RNA polymerase, known as nuclear-encoded plastid RNA polymerase (NEP), which transcribes multiple genes including housekeeping genes. The plastid genome of angiosperms contains approximately 150 genes and more than 200 transcription start sites (TSSs). Nonetheless, the number of known PEP-specific promoter regions is only 25. To clarify the genome-wide contribution of PEP in plastid transcription, we assessed the PEP-DNA binding pattern in chloroplasts by analyzing published PEP-targeted plastid-chromatin immunoprecipitation-sequencing data from 5-day-old Arabidopsis seedlings. We searched for PEP-dependent TSSs based on following four criteria: (1) strong PEP-DNA binding, (2) substantially lower PEP binding in PAP mutants, (3) presence of prokaryotic-type promoter motifs, and (4) conservation among angiosperms. We identified 32 specific PEP-binding regions which satisfy at least three criteria, including 16 regions newly suggested as PEP-associated sites in Arabidopsis. Many tRNA genes were included here, suggesting pivotal roles of PEP in tRNA expression. We also characterized 11 potential PEP-binding regions, which have PEP-binding signals but do not meet multiple criteria. Notably, some previously reported PEP-binding regions showed limited PEP binding at least under conditions we tested. Our findings provide a framework for reassessing the role of PEP to update our understanding of plastid gene expression on a genome-wide scale.