The Elongator subcomplex Elp456 is a hexameric RecA-like ATPase.
Glatt, S., Letoquart, J., Faux, C., Taylor, N.M., Seraphin, B. & Muller, C.W.
Nat Struct Mol Biol. 2012 Feb 19. doi: 10.1038/nsmb.2234.
Elongator was initially described as an RNA polymerase II-associated factor but has since been associated with a broad range of cellular activities. It has also attracted clinical attention because of its role in certain neurodegenerative diseases. Here we describe the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits each show almost identical RecA folds that form a heterohexameric ring-like structure resembling hexameric RecA-like ATPases. This structural finding is supported by different complementary in vitro and in vivo approaches, including the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification, explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of the holo-Elongator complex.
Chromatin-modifying Complex Component Nurf55/p55 Associates with Histones H3 and H4 and Polycomb Repressive Complex 2 Subunit Su(z)12 through Partially Overlapping Binding Sites.
Nowak, A.J., Alfieri, C., Stirnimann, C.U., Rybin, V., Baudin, F., Ly-Hartig, N., Lindner, D. & Muller, C.W.
J Biol Chem. 2011 Jul 1;286(26):23388-96. Epub 2011 May 5.
Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-A crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the beta-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.
Conformational flexibility of RNA polymerase III during transcriptional elongation.
Fernandez-Tornero, C., Bottcher, B., Rashid, U.J., Steuerwald, U., Florchinger, B., Devos, D.P., Lindner, D. & Muller, C.W.
EMBO J. 2010 Nov 17;29(22):3762-72. Epub 2010 Oct 22
RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 A resolution and its elongation complex at 16.5 A resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation.
Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.
Moriniere, J., Rousseaux, S., Steuerwald, U., Soler-Lopez, M., Curtet, S., Vitte, A.L., Govin, J., Gaucher, J., Sadoul, K., Hart, D.J., Krijgsveld, J., Khochbin, S., Muller, C.W. & Petosa, C.
Nature. 2009 Oct 1;461(7264):664-8.
A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.