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
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.
Subject(s)
Legionella pneumophila/enzymology , Ubiquitin-Protein Ligases/physiology , Amino Acid Sequence , Cloning, Molecular , HEK293 Cells , Humans , Legionella pneumophila/genetics , Legionnaires' Disease/genetics , Legionnaires' Disease/microbiology , Models, Molecular , Protein Conformation, alpha-Helical , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitination/geneticsABSTRACT
Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine ß-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.
Subject(s)
Cyclins/chemistry , Cyclins/metabolism , Cystathionine beta-Synthase/chemistry , Cystathionine beta-Synthase/metabolism , Amino Acid Sequence , Binding Sites/physiology , Cation Transport Proteins , Crystallization , Cyclins/genetics , Cystathionine beta-Synthase/genetics , Humans , Molecular Sequence Data , Mutation/genetics , Nucleotides/chemistry , Nucleotides/metabolism , Protein Conformation , Protein Structure, SecondaryABSTRACT
The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 A resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous alpha-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the alpha-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.
Subject(s)
Multiprotein Complexes/metabolism , Mutation/genetics , Neurodegenerative Diseases/genetics , Vesicular Transport Proteins/chemistry , Vesicular Transport Proteins/genetics , Alleles , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , HeLa Cells , Humans , Leucine/metabolism , Mice , Mice, Neurologic Mutants , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Organ Specificity , Protein Stability , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Structure-Activity Relationship , Vesicular Transport Proteins/metabolismABSTRACT
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.
Subject(s)
RNA Polymerase I , RNA Precursors , Humans , Animals , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , DNAABSTRACT
Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.
Subject(s)
RNA Polymerase III/chemistry , RNA-Directed DNA Polymerase/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Genes, Fungal , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA Polymerase III/genetics , RNA Polymerase III/metabolism , RNA, Transfer/genetics , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Retroelements , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factor TFIIIB/metabolismABSTRACT
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
Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.
Subject(s)
RNA Polymerase III/metabolism , Transcription Factor TFIIIB/metabolism , Transcription Initiation, Genetic , Amino Acid Sequence , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , DNA/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Binding , Protein Domains , Sequence Homology, Amino Acid , TATA-Box Binding Protein/chemistry , TATA-Box Binding Protein/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIIB/chemistry , Transcription Factor TFIIIB/geneticsABSTRACT
The Golgi-Associated Retrograde Protein (GARP) complex is a tethering factor involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein, we report the structural basis for the interaction of the human Ang2 subunit of GARP with the Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of the Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a binding mode in which a dityrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction.