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Cryo electron micrographs of Plastid-encoded RNA polymerase transcription elongation complex (PEP-TEC) from Sinapis alba

ABSTRACT:

SUBMITTER: Michael William Webster

PROVIDER: EMPIAR-12288 | biostudies-other |

REPOSITORIES: biostudies-other

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Structure of the plant plastid-encoded RNA polymerase.

Vergara-Cruces Ángel Á Pramanick Ishika I Pearce David D Vogirala Vinod K VK Byrne Matthew J MJ Low Jason K K JKK Webster Michael W MW

Cell 20240201 5

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-sub ...[more]

PMID: 38428394

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Cryo electron micrographs of Plastid-encoded RNA polymerase (PEP) from Sinapis alba

Project description: Not available

| EMPIAR-12279 | biostudies-other

Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from <i>Sinapis alba</i>.

Project description: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.

| S-EPMC9456514 | biostudies-literature

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

Project description: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.

2022-09-12 | PXD032739 | Pride

Mass spectrometry-based proteomic characterization of plastid-encoded RNA polymerase purified from Sinapis alba.

Project description: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. Mass spectrometry-based proteomic analysis of the obtained fraction identified the expected subunits, i.e. the core components and the PAPs as the most abundant proteins.

2022-09-12 | PXD032738 | Pride

Cryo-electron micrographs of Hantaan virus polymerase in elongation state

Project description: Not available

| EMPIAR-11541 | biostudies-other

Architecture of the spinach plastid-encoded RNA polymerase.

Project description: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.

| S-EPMC11561172 | biostudies-literature

Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba).

Project description:Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.

| S-EPMC147615 | biostudies-other

Phytochrome activates the plastid-encoded RNA polymerase for chloroplast biogenesis via nucleus-to-plastid signaling.

Project description:Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.

| S-EPMC6570650 | biostudies-literature