The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans

Abstract

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes

“…Interestingly, the incorporation of nucleotides is both faster and more promiscuous in Pol I compared with Pol II (refs. 75,76 ). The reason may lie in an evolutionarily optimized balance between speed and error rate and may reflect the unique cellular functions of each enzyme.…”

“…As shown recently, the Pol I complex contains domains and subunits that fulfil the function of separate transcription factors used by other polymerases, such as TFIIS and TFIIF (see the section 'Pol I structure and function are optimized for pre-rRNA synthesis'). For example, metazoan Pol I contains an additional domain within its largest subunit that resembles a truncated HMG box incapable of binding DNA, but this additional domain potentially provides a TOP2A docking plattform 76 . By contrast, the subunit A14 was lost from Pol I in mammals, although the overall architecture of the holoenzyme remains unchanged 76,113,192 .…”

“…For example, metazoan Pol I contains an additional domain within its largest subunit that resembles a truncated HMG box incapable of binding DNA, but this additional domain potentially provides a TOP2A docking plattform 76 . By contrast, the subunit A14 was lost from Pol I in mammals, although the overall architecture of the holoenzyme remains unchanged 76,113,192 . corresponding rDNA region, suggesting that rDNA transcription is determined at the sequence level 119 .…”

“…Pol I activity also determines rRNA quantity, and human Pol I subunit mutations are linked to at least four developmental diseases: acro facial dysostosis (Cincinnati type) 170,186 , hypomyelinating leukodystrophy (HL), Treacher Collins syndrome [187][188][189][190] and a juvenile neurodegenerative phenotype akin to the HL phenotype that is characterized by severe neurological deficits in childhood or adolescent development 191 . Mapping of these mutations to human Pol I structures allowed speculation about the molecular mechanisms underlying the pathologies 76,113,192 . Essentially, the mutations described to date are likely to cause problems in Pol I translocation during nucleotide addition, minor subunit folding defects, or intersubunit assembly or stability defects.…”