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 the 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.

Keywords: PEP associated proteins; Sinapis alba; chloroplast biogenesis; photomorphogenesis; photosynthesis; plastid-encoded RNA polymerase; transcription.

Figure 1

PEP composition and three-dimensional envelope. (a) Organelle fractionation, purification scheme and sample processing for mass spectrometry (MS/MS) or crosslinking mass spectrometry (XL/MS) or negative staining electron microscopy (eM). (b) Mass spectrometry data presented as relative iBAQ values to that of α (iBAQr) as a function of the corresponding protein coverage expressed in percentage. Subunits α, β, β’ and β” are in yellow, PAPs in blue, suspected permanent residents in black, histones in magenta and suspected purification contaminants in different shades of grey. In the shaded yellow area fall all the expected components of the PEP-A complex and correspond to the major protein mass contribution to the purified sample. (c) Sinapis alba PEP-A envelope calculated from negative staining EM acquisitions (see Figure 2 for details).

Figure 2

Negative-staining electron microscopy and 3D envelope of the PEP-A complex. (a) Overview image of the grid after negative staining. Note the homogeneity of the sample and the lack of other protein complexes. The white scale bar represents 50 nm. (b) Two-dimensional classes of PEP. (c) Three-dimensional envelope of PEP at 27.5 Å resolution calculated from 17,567 particles.

Figure 3

Mapping variable sites of the core subunits. View of the E. coli RNAP (PDB entry: 6GH5) without the ω subunit and the σ54 factor. The double-stranded DNA is colored in blue. The core subunits are drawn in spheres. (a) Mapping the variable sites as homologous in grey and nonhomologous or gaps in green. (b) Mapping only amino acid functional differences between bRNAP and PEP, as given in the sequence alignments (Figures S2–S5) The residues colored in green and orange are those displaying a strong modification of functional groups for at least 3 consecutive amino acids.

Figure 4

Phylogeny and sequence alignments of the core subunits. (a) Phylogram obtained with the Clustal Omega multi-alignment algorithm. Branch length presented as a cladogram. Major taxa included from the collection presented in the data source (Excel sheet sorted). A major incongruence from the angiosperm phylogeny tree (version IV: http://www.mobot.org accessed on 1 January 2022) is noted for Magnoliales and likely due to the study of chloroplast genes with cytoplasmic inheritance. (b) Schematic representation of the sequence context of E. coli (Ec), T. thermophilus (Tt) and A. thaliana (At) RNAP or PEP subunits as the output of a dot plot analysis performed using dotmatcher (https://www.bioinformatics.nl/cgi-bin/emboss/dotmatcher accessed on 1 January 2022). Insertions are represented in red or dashed red, with the duplicated area in pink. The splits of the bacterial β’ in the PEP β’ and β” are presented with a light-grey circle and a black triangle separating the shared regions. (c) Global alignment represented as the 11-aa rolling identity (blue) or homology (grey) percentages calculated for all taxa. In green is the 11-aa rolling identity percentage calculated in a subset of taxa corresponding to plants with detected PAPs (green). The black triangle is the evolutionary split of the rpoC gene in the rpoC1 and rpoC2 genes in the cyanobacteria. Red and blue rectangles represent dipeptides between β-β’, while yellow rectangles represent interacting peptides with PAPs, as found in the XL-MS analysis (see below).

Figure 5

Mapping protein interactions on the core complex. (a) Protein clusters determined from the XL-MS analysis (Table 1) are schematically presented with the link and scores; grey bubbles correspond to the protein not belonging to the PEP-A purified complex: RPS2, Ribosomal Protein S2; SPPA, light-inducible chloroplast protease complex associated with thylakoid membranes. Cluster 1 composed of the PAP5, FLN2, α and β’ subunits. Cluster 2 composed of PAP1, 2 and 11. (b) Model of the PEP core complex from A. thaliana built from the α, β, β’ and β” subunits modelized using AlphaFold [42] and superimposed onto the E. coli RNAP catalytic core and colored as follows: α subunit in red, β subunit in pink, β’ subunit in yellow and β” in green. The van der Waals spheres display the peptides of the α and β’ subunits that are nearby to PAP5 and FLN2 (Table 1). (c) View of the catalytic core from the E. coli RNAP (PDB entry: 6GH5 [40]).