Synthesis of the ribosomal RNA precursor in human cells: mechanisms, factors and regulation.

The ribosomal RNA precursor (pre-rRNA) comprises three of the four ribosomal RNAs and is synthesized by RNA polymerase (Pol) I. Here, we describe the mechanisms of Pol I transcription in human cells with a focus on recent insights gained from structure-function analyses. The comparison of Pol I-specific structural and functional features with those of other Pols and with the excessively studied yeast system distinguishes organism-specific from general traits. We explain the organization of the genomic rDNA loci in human cells, describe the Pol I transcription cycle regarding structural changes in the enzyme and the roles of human Pol I subunits, and depict human rDNA transcription factors and their function on a mechanistic level. We disentangle information gained by direct investigation from what had apparently been deduced from studies of the yeast enzymes. Finally, we provide information about how Pol I mutations may contribute to developmental diseases, and why Pol I is a target for new cancer treatment strategies, since increased rRNA synthesis was correlated with rapidly expanding cell populations.

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

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.

Snapshots of RNA polymerase III in action - A mini review.

RNA polymerase (Pol) III is responsible for the transcription of tRNAs, 5S rRNA, U6 snRNA, and other non-coding RNAs. Transcription factors such as TFIIIA, -B, -C, SNAPc, and Maf1 are required for promoter recognition, promoter opening, and Pol III activity regulation. Recent developments in cryo-electron microscopy and advanced purification approaches for endogenous multi-subunit complexes accelerated structural studies resulting in detailed structural insights which allowed an in-depth understanding of the molecular mechanisms underlying Pol III transcription. Here, we summarize structural data on Pol III and its regulating factors providing a three-dimensional framework to guide further analysis of RNA polymerase III.

Cytosine base modifications regulate DNA duplex stability and metabolism.

DNA base modifications diversify the genome and are essential players in development. Yet, their influence on DNA physical properties and the ensuing effects on genome metabolism are poorly understood. Here, we focus on the interplay of cytosine modifications and DNA processes. We show by a combination of in vitro reactions with well-defined protein compositions and conditions, and in vivo experiments within the complex networks of the cell that cytosine methylation stabilizes the DNA helix, increasing its melting temperature and reducing DNA helicase and RNA/DNA polymerase speed. Oxidation of methylated cytosine, however, reverts the duplex stabilizing and genome metabolic effects to the level of unmodified cytosine. We detect this effect with DNA replication and transcription proteins originating from different species, ranging from prokaryotic and viral to the eukaryotic yeast and mammalian proteins. Accordingly, lack of cytosine methylation increases replication fork speed by enhancing DNA helicase unwinding speed in cells. We further validate that this cannot simply be explained by altered global DNA decondensation, changes in histone marks or chromatin structure and accessibility. We propose that the variegated deposition of cytosine modifications along the genome regulates DNA helix stability, thereby providing an elementary mechanism for local fine-tuning of DNA metabolism.

Conserved strategies of RNA polymerase I hibernation and activation.

RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms 'activation by cleft contraction' and 'hibernation by dimerization' are unique to the regulation of this enzyme, but structure-function analysis is limited to baker's yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the 'connector' domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.

Structure of human RNA polymerase III.

In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.