Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba

Abstract

RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.

Architecture of the spinach plastid-encoded RNA polymerase

“…These findings suggest that PAP1 may be involved in the transcription initiation process of PEP. Although PAP2 was not identified in our structure, cross-linking mass spectrometry detected interactions between PAP1 and PAP2 in the PEP complex purified from Arabidopsis , implying that PAP1 and PAP2 may function together 28 . PAP11 specifically positioned at the forefront of the upper arm adjacent to the β’ subunit.…”

Chloroplast gene expression: Recent advances and perspectives

“…Twelve PAPs have been identified in Arabidopsis chloroplasts, PAP1 to PAP12 ( Steiner et al., 2011 ; Pfannschmidt et al., 2015 ). These PAPs bind specifically and tightly to the Rpo catalytic core, forming well-conserved protrusions as in other RNA polymerases, although the 3D map resolution of the PEP complex is currently insufficient to determine the relative positions of these PAPs within the PEP complex ( Ruedas et al., 2022 ). All these PAPs are necessary for chloroplast transcription and show a low level of functional redundancy.…”

pTAC3 and pTAC14 are required for binding of plastid-encoded RNA polymerase to DNA

“…The molecular architecture of the PEP complex (Ruedas et al 2022) and functions of individual PAPs remain only partially understood (Pfannschmidt et al 2015).…”

“…Arabidopsis thaliana has 12 PAPs, which are of eukaryotic origin and are encoded in the nuclear genome. The molecular architecture of the PEP complex (Ruedas et al 2022) and functions of individual PAPs remain only partially understood (Pfannschmidt et al 2015).…”