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

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.Funding: This work was funded by the Intramural Research Program of the National Institutes of Health (to MPM)(Project Number: 1ZIAHD008893-07) and by the Spanish Ministry of Economy and Competitiveness Grant (to AH)(BFU2014-59759-R) and the Severo Ochoa Excellence Accreditation (to AH)(SEV-2016-0644). This study made use of the Diamond Light Source beamline I04 (Oxfordshire, UK) and ALBA synchrotron beamline BL13-XALOC, funded in part by the Horizon 2020 programme of the European Union, iNEXT (H2020 Grant # 653706). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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

The Legionella pneumophila effector RavN is an E3 ligase that hijacks host-cell ubiquitination

Trabajo presentado en el Workshop Contribution of bacterial injection systems to human disease, celebrado en Jaén (España) del 05 al 07 de noviembre de 2018

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

The N-terminal region of RavN mediates E3 ligase activity.

<p>(A) Shown here is the schematic representation of the RavN fragments examined in the <i>in vitro</i> ubiquitylation assay in (B). The degree of sequence identity of the N-terminal E3 ligase domain and C-terminal region (CTD) among <i>Legionella</i> RavN homologs (as seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.s002" target="_blank">S2A Fig</a>) is indicated [in %]. (B) The <i>in vitro</i> ubiquitylation assay of RavN truncation variants was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.g001" target="_blank">Fig 1D</a> using UbcH5a as E2. The formation of poly-ubiquitylated species was detected with HRP-conjugated streptavidin.</p

RavN has ubiquitin ligase activity.

<p>(A) The silver-stained SDS-PAGE gel showing proteins present on either uncoated control beads (left) or His₆-RavN-coated beads before (right) or after (center) incubation with lysate from HEK293T cells. The protein bands corresponding to His₆-RavN or mono-ubiquitylated His₆-RavN are indicated with arrows. (B) His₆-tagged RavN, but not RavI or AnkJ, is ubiquitylated upon incubation with HEK293T cell lysate. Top: Poly-ubiquitylated species (poly-ub) detected by immunoblot using anti-ubiquitin antibody. Bottom: Total amount of His₆-tagged effector proteins present in each lane. (C) FLAG-RavN is poly-ubiquitylated in transiently transfected HEK293T cells. FLAG-tagged RavN was precipitated from HEK293T cell lysate, and ubiquitylation (poly-ub) was detected by immunoblot using anti-ubiquitin antibody. Total amount of FLAG-RavN was detected by anti-FLAG antibody shown at the bottom. (D) <i>In vitro</i> ubiquitylation assay. Purified recombinant GST-RavN was incubated in the presence of a panel of mammalian E2s or without E2s (no E2), and the formation of poly-ubiquitylated species (poly-ub) was detected using HRP-conjugated streptavidin.</p

The U-box-E2 binding interface is conserved in RavN.

<p>(A) Superimposition of RavN_1-55 (orange) and two related E3s (RING domain of Ark2C, light green; PDB 5D0K; U-box domain of CHIP, dark pink, PDB 2OXQ) in complex with their respective E2s (UbcH5b and UbcH5a) shown in gray. A close-up view highlights residues in RavN that are homologous to those located at the core of the E2 binding site in the other two E3s. (B) Residues conserved in all ten RavN homologs (labeled with an asterisk in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.s002" target="_blank">S2A Fig</a>) cluster into two patches at the surface of the RavN_1-123 molecule (indicated by ovals), one of which overlaps with the proposed E2 binding interface. (C) The effect of interface residue substitutions in RavN was determined in the <i>in vitro</i> reconstitution assay using UbcH5a as E2. The formation of poly-ubiquitylated RavN was detected after 5, 10, 30, and 60 minutes with HRP-conjugated streptavidin. The total amount of GST-RavN in each reaction was determined by immunoblot using anti-GST antibody. WT: wild-type RavN. (D) Cell-based ubiquitylation assay. FLAG-tagged RavN variants simultaneously produced with HA-tagged ubiquitin in HEK293T cells were immuno-precipitated, and poly-ubiquitylation was detected by immunoblot using anti-HA antibody (right). Total amount of FLAG-tagged RavN or RavN variants was detected by immunoblot using anti-FLAG antibody (left). (E) Inhibition of yeast growth is dependent on the E3 ligase activity of RavN. <i>S</i>. <i>cerevisiae</i> INVSc1 cells containing plasmids encoding wild-type (WT) RavN or the indicated RavN variants were grown in the presence of either glucose (to repress <i>ravN</i> expression) or galactose (induces <i>ravN</i> expression), and cell growth was determined after 48 hours by spotting serial dilutions.</p

The E3 ligase domain of RavN has a contorted U-box fold.

<p>(A) Schematic representation of the structure of RavN_1-123 as ribbon diagram displayed in two orientations (rotated by 90° along the <i>x</i> axis). Secondary elements are indicated as spirals (helices) or arrows (beta strands), with the RING/U-box motif colored in orange and the C-terminal structure colored in slate. (B) Topology diagram of RavN_1-123 with the same color scheme as shown in (A). Numbers indicate amino acid residues. (C) Superimposition of the U-box domain of RavN (RavN_1-55, orange) with the RING domains of Ark2C (PDB 5D0I, light green) and RNF146 (PDB 4QPL, light blue), and the U-box domain of NleG (PDB 2KKX, light pink). Conserved structural elements shared among RING/U-box domains are labeled as L1_E3, L2_E3, αA, βA, and βB. The position of αA is occupied by loop L4 in RavN (L4_RavN). (D) Topology diagrams of the structural homologs of RavN. The same color scheme is used as in (C) and in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.g004" target="_blank">Fig 4A</a>. The residues located at the E2 binding interface in RavN, Ark2C, and CHIP (as seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.g004" target="_blank">Fig 4A</a>) are indicated.</p

The flexible α3 helix is not required for the E3 ligase activity of RavN.

<p>(A) Each asymmetric unit within the protein crystal contained a RavN_1-123 molecule (shown as red, slate and green ribbon diagrams) that differed with respect to the orientation of their helix α3 (α3_A, α3_B, and α3_C). (B) Superimposition of the three conformations of RavN_1-123 found in the asymmetric unit. Structures are presented as ribbon diagrams, with the overlaid part colored in gray and the variable α3 helix colored in red, slate and green. (C) Top: Schematic representation of RavN fragments examined in the <i>in vitro</i> reconstitution assay (bottom) using UbcH5a as E2. Poly-ubiquitylated RavN was detected with HRP-conjugated streptavidin, and the total amount of GST-RavN present in each reaction was confirmed by immunoblot using anti-GST antibody. (D) Phe103 is important for E3 ligase activity of RavN. An <i>in vitro</i> reconstitution assay was used to determine the effect of the indicated amino acid substitutions within region 101–110 on the E3 ligase activity of RavN^L43S/P47S. UbcH5a was added as E2 enzyme. The formation of poly-ubiquitin species was detected using HRP-conjugated streptavidin. The total amount of GST-RavN in each lane was determined by immunoblot using anti-GST antibody. (E) The position of Phe103 relative to the three E2 binding interface residues Ile8, Leu43, and Pro47 in RavN is shown. Color scheme as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006897#ppat.1006897.g003" target="_blank">Fig 3A</a>.</p