Architecture of the spinach plastid-encoded RNA polymerase

Abstract

The plastid-encoded RNA polymerase serves as the principal transcription machinery within chloroplasts, transcribing over 80% of all primary plastid transcripts. This polymerase consists of a prokaryotic-like core enzyme known as the plastid-encoded RNA polymerase core, and is supplemented by newly evolved associated proteins known as PAPs. However, the architecture of the plastid-encoded RNA polymerase and the possible functions of PAPs remain unknown. Here, we present the cryo-electron microscopy structure of a 19-subunit plastid-encoded RNA polymerase complex derived from spinach (Spinacia oleracea). The structure shows that the plastid-encoded RNA polymerase core resembles bacterial RNA polymerase. Twelve PAPs and two additional proteins (FLN2 and pTAC18) bind at the periphery of the plastid-encoded RNA polymerase core, forming extensive interactions that may facilitate complex assembly and stability. PAPs may also protect the complex against oxidative damage and has potential functions in transcriptional regulation. This research offers a structural basis for future investigations into the functions and regulatory mechanisms governing the transcription of plastid genes.

Fig. 1. Overall architecture of the Spinacia oleracea (Sp.) PEP complex.

Different views show the overall arrangement of the subunits in the Sp. PEP complex. The subunits are shown as cartoons and colored individually as indicated. α, RNA polymerase α subunit; β, RNA polymerase β subunit; β', RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP4, iron superoxide dismutase 3 (FSD3); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP6, fructokinase-like protein 1 (FLN1); PAP7, plastid transcriptionally active chromosome protein 14 (pTAC14); PAP8, plastid transcriptionally active chromosome protein 6 (pTAC6); PAP9, iron superoxide dismutase 2 (FSD2); PAP10, thioredoxin Z (Trx Z); PAP11, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase MurE homolog; PAP12/ω, plastid transcriptionally active chromosome protein 7 (pTAC7); PAP13, fructokinase-like protein 2 (FLN2); PAP14, plastid transcriptionally active chromosome protein 18 (pTAC18). Among these subunits, the two copies of the α subunit are labeled as α1 and α2 and given different colors, while the two copies of the PAP10 subunit are labeled as PAP10₁ and PAP10₂ and colored the same due to their different locations.

Fig. 2. Comparison of the Spinacia oleracea (Sp.) PEP core RNA polymerase and bacterial RNAP.

Overall structural similarity between the Sp. PEP core polymerase (a), Synechocystis sp. PCC 6803 (Syn) RNAP (PDB 8GZG) (b) and E. coli RNAP (PDB 6GH5) (c). d Superimposition of the structures for the Sp. PEP core polymerase and Syn RNAP (root mean square deviation (r.m.s.d.) of 1.907 Å for Cα atoms by PyMOL) showing that the Sp. PEP core polymerase adopts the same folds as Syn RNAP. e–g Structural comparison of the key structural domains (clamp, protrusion, lobe, flap, and jaw) between the Sp. PEP core polymerase and Syn RNAP. h Structural comparison of the key structural motifs in the active site (catalytic loop, trigger loop, bridge helix, and rim helices) between the Sp. PEP core polymerase and Syn RNAP. α1, α2, RNA polymerase α subunit; β, RNA polymerase β subunit; β', RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP12/ω, plastid transcriptionally active chromosome protein 7 (pTAC7); TL, trigger loop; BH, bridge helix; β''-RH, β'' rim helices; β'CH, clamp helices; SI3, the β'' arch domain of sequence insertion 3.

Fig. 3. Comparison of the β'' subunit of the Spinacia oleracea (Sp.) PEP core polymerase and cyanobacterial RNAP.

a Comparison of the β'' subunit of the Sp. PEP core polymerase and Synechocystis sp. PCC 6803 (Syn) RNAP (PDB 8GZG) (r.m.s.d. of 5.702 Å for Cα atoms by PyMOL). b Comparison of the β'' arch domain of sequence insertion 3 (SI3) in the Sp. PEP core polymerase and Syn RNAP (r.m.s.d. of 2.965 Å for Cα atoms by PyMOL, amino acids [aa] 340–1131). The PEP β'' SI3 arch shows a larger fin that provides a binding site for PAPs. c Comparison of the region of β'' other than SI3 between the Sp. PEP core polymerase and Syn RNAP (r.m.s.d. of 1.137 Å for Cα atoms by PyMOL). d Close-up view of the jaw domain in Syn RNAP and the corresponding position in the PEP core polymerase. The dashed line represents the unique domain of PAP1 connecting residues E544 and R721, which occupies the position corresponding to the jaw in Syn RNAP. e Close-up view of the βαβ motif adjacent to the secondary channel in the Sp. PEP core polymerase and Syn RNAP. The βαβ motif in the Sp. PEP core polymerase contributes to the binding site for PAP7 and PAP8. β'', RNA polymerase β'' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP4, iron superoxide dismutase 3 (FSD3); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP6, fructokinase-like protein 1 (FLN1); PAP7, plastid transcriptionally active chromosome protein 14 (pTAC14); PAP8, plastid transcriptionally active chromosome protein 6 (pTAC6); PAP9, iron superoxide dismutase 2 (FSD2); PAP14, plastid transcriptionally active chromosome protein 18 (pTAC18). SI3, the β'' arch domain of sequence insertion 3; BH, bridge helix; β''-RH, β'' rim helices.

Fig. 4. Comparison of the β and β' subunits of the Spinacia oleracea (Sp.) PEP core polymerase and cyanobacterial RNAP.

a Structural superimposition of the β' subunit between the Sp. PEP core polymerase and Synechocystis sp. PCC 6803 (Syn) RNAP (PDB 8GZG) (r.m.s.d. of 1.257 Å for Cα atoms by PyMOL). The conserved active site is highlighted in dark blue. Close-up view of the β' SI1 and the β' blade domain (b), the β' N terminus and β'CH (c), and the toe of the clamp (d). Structural domains showing a change in conformation are highlighted in magenta. e Structural superimposition of the β subunit between the Sp. PEP core polymerase and Syn RNAP (r.m.s.d. of 1.280 Å for Cα atoms by PyMOL). f Close-up view of two loops close to the β lobe domain of PEP, labeled as loop 1 and loop 2. The two loops adopt different conformation compared to those of Syn RNAP (right). The two loops are highlighted in orange. β, RNA polymerase β subunit; β', RNA polymerase β' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP8, plastid transcriptionally active chromosome protein 6 (pTAC6); PAP11, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase MurE homolog; β'-SI1, a sequence insertion in β' (residues 545–581); β'CH, clamp helices.

Fig. 5. Arrangement of PAPs in the Spinacia oleracea (Sp.) PEP complex.

Position of PAPs located on the upper arm and the lower arm (a) and the intersection of the two arms (b). All PAPs are presented as cartoons. The PAPs are colored individually as indicated in Fig. 1; the Sp. PEP core subunits are colored in gray. c Summary table showing that the PAPs interact extensively with the PEP core subunits and with each other. α1, α2, RNA polymerase α subunit; β, RNA polymerase β subunit; β', RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP4, iron superoxide dismutase 3 (FSD3); PAP6, fructokinase-like protein 1 (FLN1); PAP7, plastid transcriptionally active chromosome protein 14 (pTAC14); PAP8, plastid transcriptionally active chromosome protein 6 (pTAC6); PAP9, iron superoxide dismutase 2 (FSD2); PAP10₁, PAP10₂, thioredoxin Z (Trx Z); PAP11, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase MurE homolog; PAP12/ω, plastid transcriptionally active chromosome protein 7 (pTAC7); PAP13, fructokinase-like protein 2 (FLN2); PAP14, plastid transcriptionally active chromosome protein 18 (pTAC18).

Fig. 6. PAP1, PAP7, and PAP11 stabilize the Spinacia oleracea (Sp.) PEP core polymerase through embracing the clamp-claw.

a PAP1, PAP7, and PAP11 (shown as surface presentation) embrace the clamp-claw of the Sp. PEP core polymerase. b Detailed interactions of the N-terminal pentatricopeptide repeats (PPRs) of PAP1 (PAP1-PPR_N) and the C-terminal PPRs of PAP1 (PAP1-PPR_C) with the β, β', and β'' subunits of the Sp. PEP core polymerase. c Detailed interactions of the PAP1-unique domain (aa 394–782) located between PAP1-PPR_N and PAP1-PPR_C with the β'-clamp and β'' of the Sp. PEP core polymerase. d Detailed interactions of PAP7 with the Sp. PEP core polymerase subunits (β'', PAP12/ω), PAP1, and PAP8. e Detailed interactions of PAP11 with the β‘ clamp and PAP1, and structural comparison of PAP11 and E. coli MurE (PDB 1E8C) (r.m.s.d. of 3.226 Å for Cα atoms by PyMOL). β, RNA polymerase β subunit; β‘, RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP7, plastid transcriptionally active chromosome protein 14 (pTAC14); PAP11, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase MurE homolog; PAP12/ω, plastid transcriptionally active chromosome protein 7 (pTAC7); β''-RH, rim helices; SI3, the β'' arch domain of sequence insertion 3; β'CH, clamp helices.

Fig. 7. PAP5 and PAP8 contribute to assembly and stability of the Spinacia oleracea (Sp.) PEP core polymerase.

a, b Structures and locations of PAP5 and PAP8 shown from different views. c–f Detailed views of the interactions between PAP5 and other subunits. g, h Detailed views of the interactions between PAP8 and other subunits shown from different views. α1, α2, RNA polymerase α subunit; β, RNA polymerase β subunit; β', RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP6, fructokinase-like protein 1 (FLN1); PAP7, plastid transcriptionally active chromosome protein 14 (pTAC14); PAP8, plastid transcriptionally active chromosome protein 6 (pTAC6); PAP10₁, thioredoxin Z (Trx Z); PAP12/ω, plastid transcriptionally active chromosome protein 7 (pTAC7); BH, bridge helix; β''-RH, rim helices; SI3, the β'' arch domain of sequence insertion 3; β'-SI1, a sequence insertion in β' (residues 545–581).

Fig. 8. PAP3, PAP14, PAP4, and PAP9 scaffold the lobe-protrusion-claw.

a, b Structure and locations of PAP3, PAP14, PAP4, and PAP9 shown from different views. PAP3, PAP14, PAP4, and PAP9 are shown and colored as indicated in Fig. 1. The β subunit is shown as a gray cartoon. To show its detailed interactions with the PEP core subunits and other PAPs, PAP3 was divided into three domains: the short N-terminal domain (PAP3-NTD; aa 72–150), the middle domain (PAP3-MD; aa 160–380) and the C-terminal domain (PAP3-CTD; aa 381–618). c–e Detailed view of the interactions between PAP3-NTD, PAP3-MD, and PAP3-CTD with PEP SI3, β''-RH, PAP14, PAP4, and PAP9. PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP4, iron superoxide dismutase 3 (FSD3); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP9, iron superoxide dismutase 2 (FSD2); PAP14, plastid transcriptionally active chromosome protein 18 (pTAC18); SI3, the β'' arch domain of sequence insertion 3; β“-RH, rim helices.

Fig. 9. Redox clusters of the PAP6–PAP10₁ and PAP13–PAP10₂ heterodimers.

a Structure and locations of the PAP6–PAP10₁ and PAP13–PAP10₂ heterodimers shown from different views. b Detailed view of the interactions between the PAP6–PAP10₁ heterodimer and the Spinacia oleracea (Sp.) PEP core polymerase and other PAPs. The black arrow highlights the PAP10₁-specific extended loop absent in Trx M, which interacts with PAP5 and the PEP β subunit. The red arrows highlight the folds of PAP6 that are distinctly unlike those in V. cholerae fructokinase (FRK) and mediate the interactions with PEP core subunits and other PAPs. c Structural details of the interactions between the PAP13–PAP10₂ heterodimer and the α-homodimer of the Sp. PEP core polymerase. α1, α2, RNA polymerase α subunit; PAP3, plastid transcriptionally active chromosome protein 10 (pTAC10); PAP5, plastid transcriptionally active chromosome protein 12 (pTAC12/HEMERA); PAP6, fructokinase-like protein 1 (FLN1); PAP10₁, PAP10₂, thioredoxin Z (Trx Z); PAP13, fructokinase-like protein 2 (FLN2); SI3, the β'' arch domain of sequence insertion 3; BH, bridge helix.

Fig. 10. Models of active chloroplast transcription complexes.

a Model of an open, initially transcribing complex of PEP (PEPitc) shown from different views. The model was constructed by superimposing an AlphaFold2 model of the Spinacia oleracea (Sp.) σ factor (SigF) and the Sp. PEP complex with Synechocystis sp. PCC 6803 (Syn) RPitc (PDB 8GZG). The nucleic acid molecule was positioned based on structural superposition with Syn RPitc (PDB 8GZG). The PEP core is shown as a cartoon in gray and PAPs are shown as surfaces in the same color as in Fig. 1. The nucleic acids are shown as cartoons. Both SigF and DNA can be accommodated without large conformational rearrangements or loss of factors in PEPitc. b Close-up view of the active site in the modeled PEPitc. The active site is shown as a blue surface. The bridge helix (BH) and trigger loop (TL) are shown as cartoons. SigF and nucleic acids can be accommodated without major clashes. c Close-up view of the minor clash between β'CH and the downstream DNA in PEPitc. d Model of an actively elongating complex of PEP (PEP EC). The model was constructed by superimposing the Sp. PEP complex with the Syn EC (PDB 8SYI). The nucleic acid molecule was positioned based on structural superimposition with the Syn EC (PDB 8SYI). No large conformational rearrangements or loss of factors are required to form the PEP EC. e Close-up view of the active site in the modeled PEP EC. The clash is identical to that seen with the PEPitc in c. β', RNA polymerase β' subunit; β'', RNA polymerase β'' subunit; PAP1, plastid transcriptionally active chromosome protein 3 (pTAC3); PAP11, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase MurE homolog; SI3, the β'' arch domain of sequence insertion 3; BH, bridge helix; TL, trigger loop; β'CH, clamp helices.