Characterization vy crosslinking-mass spectrometry of plastid-encoded RNA polymerase purified from Sinapis alba.

RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Crosslinking coupled to mass spectrometry provided initial structural information about the relative position of PEP subunits within the complex.