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

Structural basis of RNA polymerase III transcription initiation.

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

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

ABSTRACTRetrotransposons are endogenous elements that have the ability to mobilise their DNA and integrate at different locations in the host genome. In budding yeast, the Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase III-transcribed genes, such as the genes of transfer RNAs, representing a paradigm for specific targeted integration.Here we present the cryo-EM reconstruction at 4.0 Å-resolution of an active Ty3 strand-transfer complex (Ty3 intasome) caught in the act of integrating onto a specific tRNA gene bound to the RNA Polymerase III general transcription factor TFIIIB, which is required for Ty3 specific targeting.The structure unravels the molecular mechanisms underlying Ty3 integration specificity at RNA Polymerase III-transcribed genes and sheds light into the architecture of a retrotransposon integration machinery during the process of strand transfer at a genomic locus. The Ty3 intasome establishes contacts with a region of the TATA-binding protein (TBP), a subunit of TFIIIB, which is blocked by the ubiquitous transcription regulator negative cofactor 2 (NC2) in RNA Pol II-transcribed genes.A previously unrecognised chromodomain of the Ty3 integrase mediates non- canonical interactions with TFIIIB and the tRNA gene itself, defining with extreme precision the position of the integration site. Surprisingly, Ty3 retrotransposon tethering to TFIIIB topologically resembles LEDGF/p75 transcription factor targeting by HIV retrovirus, highlighting mechanisms of convergent evolution by unrelated mobile elements and host organisms.The Ty3 intasome-TFIIIB-tRNA promoter complex presented here represents a detailed molecular snapshot of a general transcription factor’s co-option by a mobile element, resulting in harmless integration into the host genome.</jats:p

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

AbstractRetrotransposons 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.</jats:p

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein Complex with the t-SNARE Syntaxin 6

SummaryThe 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

Structural characterization of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases

Resumen del trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018.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 lacks the central alpha helix commonly found in other U-box domains of eukaryotic E3s. These structural characteristics indicate 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 thatL. pneumophilais dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.

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

Structure of human RNA Polymerase III

ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.</jats:p

Structure of human RNA polymerase III

AbstractIn 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.</jats:p