{"database":"iProX","file_versions":[],"scores":null,"additional":{"omics_type":["Proteomics"],"submitter":["Jie Li"],"species":["Methanococcus Maripaludis X1"],"full_dataset_link":["http://www.iprox.org/page/project.html?id=IPX0018596000"],"submitter_email":["dhp2020191498@163.com"],"submitter_affiliation":["Institute of Microbiology, Chinese Academy of Sciences"],"sample_protocol":[""],"repository":["iProX"],"data_protocol":[""],"pubmed_abstract":["Archaeal transcription is driven by a multi-subunit RNA polymerase (RNAP) structurally and functionally homologous to eukaryotic RNAP II. However, its <i>in vivo</i> biogenesis remains uncharacterized. Here, we present the first comprehensive dissection of archaeal RNAP assembly using <i>Methanococcus maripaludis</i> as a genetically tractable model. Through an integrative strategy combining size exclusion chromatography, native purification, Western blotting, mass spectrum, and <i>in vitro</i> disassembly, we delineate a modular and hierarchical assembly pathway comprising five major subcomplexes. A stable Rpo3-11-2'2″ subcomplex was resolved by <i>in vitro</i> disassembly analysis, supporting a directional assembly route. Quantitative analyses revealed that RNAP subunits are maintained at balanced intracellular levels, and disruption of this stoichiometry, particularly overexpression of small subunits Rpo12 and Rpo11, disrupted RNAP assembly, reduced holoenzyme formation, and impaired growth. Rpo12 overexpression was associated with increased Rpo3/12 subcomplex, whereas Rpo11 overexpression caused accumulation of an Rpo3-11-10 intermediate; both coincided with reduced downstream assembly and holoenzyme formation, underscoring the importance of balanced subunit abundance for productive RNAP assembly. Moreover, mass spectrum identified chaperonin Hsp60 consistently co-eluting with RNAP subcomplexes, implying it as a candidate assembly cofactor. Together, these findings establish an <i>in vivo</i> framework for archaeal RNAP assembly, revealing a modular and hierarchical biogenesis pathway governed by stoichiometry-sensitive checkpoints. Given the shared subcomplex architecture and assembly logic, these insights not only fill a longstanding knowledge gap in archaeal RNAP assembly but also illuminate its evolutionary continuity with bacterial and eukaryotic systems and uncover regulatory vulnerabilities with potential applied relevance.IMPORTANCERNA polymerase is the core transcriptional machinery that underpins gene expression across domains of life, yet the <i>in vivo</i> assembly of the multi-subunit RNAP in archaea has remained poorly understood. This study provides the first <i>in vivo</i> dissection of archaeal RNA polymerase assembly, uncovering a modular and evolutionarily conserved pathway whose progression depends on balanced subunit stoichiometry. Imbalance of small subunits selectively disrupts early assembly steps, limits holoenzyme formation, and compromises cellular growth, establishing assembly fidelity as an intrinsic regulatory layer of archaeal transcription. By defining conserved assembly logic and stoichiometry-dependent checkpoints, this work fills a major gap in archaeal transcription biology, uncovers mechanistic parallels with bacterial and eukaryotic systems, and highlights RNA polymerase assembly as a previously unrecognized vulnerability in methanogenic archaea with potential relevance to methane mitigation."],"pubmed_title":["Dissection of &lt;i&gt;in vivo&lt;/i&gt; archaeal RNA polymerase assembly reveals a modular pathway and stoichiometric control."],"pubmed_authors":["Li Jie J, Qi Wen W, Duan Huaping H, Ren Derong D, Liu Jingfang J, Dong Xiuzhu X"],"additional_accession":[]},"is_claimable":false,"name":"Dissection of In Vivo Archaeal RNA Polymerase Assembly Reveals a Modular Pathway and Stoichiometric Control","description":"These findings establish a modular and hierarchical framework for archaeal RNAP assembly, uncover key regulatory checkpoints, and demonstrate the essential role of small subunit stoichiometry. Given the shared subcomplex architecture and assembly logic, these insights not only fill a longstanding knowledge gap in archaeal RNAP assembly but also illuminate its evolutionary continuity with bacterial and eukaryotic systems and uncover regulatory vulnerabilities with potential applied relevance.","dates":{"publication":"Mon Jul 27 00:00:00 GMT+01:00 2026"},"accession":"PXD081683","cross_references":{"TAXONOMY":["1053692"],"pubmed":["42517641"]}}