ABSTRACT
RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4-4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.
Subject(s)
RNA Polymerase III/metabolism , RNA Polymerase III/ultrastructure , Transcription Initiation, Genetic , Cryoelectron Microscopy , DNA/chemistry , DNA/metabolism , DNA/ultrastructure , Models, Molecular , Nucleic Acid Conformation , Promoter Regions, Genetic , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA Polymerase I/chemistry , RNA Polymerase II/chemistry , RNA Polymerase III/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Templates, Genetic , Transcription Factor TFIIIB/chemistry , Transcription Factor TFIIIB/metabolism , Transcription Factor TFIIIB/ultrastructure , Transcription Factors, TFII/chemistryABSTRACT
Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.
Subject(s)
Cerebellar Diseases/metabolism , Endonucleases/metabolism , Mutation , RNA Precursors/metabolism , RNA Splicing , RNA, Transfer/metabolism , Animals , Cerebellar Diseases/genetics , Crystallography, X-Ray , Endonucleases/chemistry , Endonucleases/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Endoribonucleases/metabolism , HEK293 Cells , Humans , Introns/genetics , Protein Conformation , Protein Multimerization , RNA Precursors/genetics , RNA, Transfer/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sf9 Cells , SpodopteraABSTRACT
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.
Subject(s)
RNA Polymerase III/chemistry , Cryoelectron Microscopy , DNA-Directed RNA Polymerases/genetics , Enzyme Stability , HeLa Cells , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Subunits , RNA Polymerase III/genetics , RNA Polymerase III/metabolism , Scattering, Small Angle , X-Ray DiffractionABSTRACT
RNA polymerase III catalyses the synthesis of tRNAs in eukaryotic organisms. Through combined biochemical and structural characterisation, multiple auxiliary factors have been identified alongside RNA Polymerase III as critical in both facilitating and regulating transcription. Together, this machinery forms dynamic multi-protein complexes at tRNA genes which are required for polymerase recruitment, DNA opening and initiation and elongation of the tRNA transcripts. Central to the function of these complexes is their ability to undergo multiple conformational changes and rearrangements that regulate each step. Here, we discuss the available biochemical and structural data on the structural plasticity of multi-protein complexes involved in RNA Polymerase III transcriptional initiation and facilitated re-initiation during tRNA synthesis. Increasingly, structural information is becoming available for RNA polymerase III and its functional complexes, allowing for a deeper understanding of tRNA transcriptional initiation. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.