ABSTRACT
The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.
Subject(s)
Histone-Lysine N-Methyltransferase , Intracellular Signaling Peptides and Proteins , Myeloid-Lymphoid Leukemia Protein , Nucleosomes , Ubiquitination , Cryoelectron Microscopy , Histone-Lysine N-Methyltransferase/chemistry , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/genetics , Nucleosomes/enzymology , Protein BindingABSTRACT
Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.
Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Animals , Catalytic Domain , Chromatin/metabolism , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/ultrastructure , Histones/chemistry , Histones/genetics , Humans , Methylation , Models, Molecular , Nucleosomes/metabolism , Protein Processing, Post-Translational , Receptor Cross-Talk , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitination , Xenopus laevisABSTRACT
The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.
Subject(s)
Fungal Proteins/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Mutation , Protein Binding , Protein Conformation , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III.
Subject(s)
DNA/metabolism , RNA Polymerase III/biosynthesis , HEK293 Cells , Humans , Protein Structure, SecondaryABSTRACT
RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.
Subject(s)
DNA/chemistry , Protein Subunits/chemistry , RNA Polymerase I/chemistry , RNA/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Gene Expression , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Folding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , RNA/genetics , RNA/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/isolation & purification , RNA Polymerase I/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Here, we discuss the overall architecture of the RNA polymerase I (Pol I) and III (Pol III) core enzymes and their associated general transcription factors in the context of models of the Pol I and Pol III pre-initiation complexes, thereby highlighting potential functional adaptations of the Pol I and Pol III enzymes to their respective transcription tasks. Several new insights demonstrate the great degree of specialization of each of the eukaryotic RNA polymerases that is only beginning to be revealed as the structural and functional characterization of all eukaryotic RNA polymerases and their pre-initiation complexes progresses.
Subject(s)
Binding Sites , Conserved Sequence , Multiprotein Complexes/metabolism , RNA Polymerase III/metabolism , RNA Polymerase I/metabolism , Transcription Initiation, Genetic , Promoter Regions, Genetic , Protein Binding , Protein Interaction Domains and Motifs , RNA Polymerase I/chemistry , RNA Polymerase III/chemistry , Substrate Specificity , Transcription Factor TFIIB/metabolism , Transcription Factors, TFII/metabolismABSTRACT
Electron cryomicroscopy reconstructions of elongating RNA polymerase (Pol) III at 3.9 Å resolution and of unbound Pol III (apo Pol III) in two distinct conformations at 4.6 Å and 4.7 Å resolution allow the construction of complete atomic models of Pol III and provide new functional insights into the adaption of Pol III to fulfill its specific transcription tasks.
Subject(s)
RNA Polymerase III/chemistry , Transcription, Genetic , Cryoelectron Microscopy , Humans , Models, Molecular , RNA Polymerase III/antagonists & inhibitors , RNA Polymerase III/metabolism , RNA Polymerase III/ultrastructureABSTRACT
The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.
Subject(s)
Chaetomium/enzymology , DNA Helicases/chemistry , DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , Catalytic Domain , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82-C34-C31 heterotrimer in close proximity to the stalk. The C53-C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.
Subject(s)
Models, Molecular , RNA Polymerase III/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Cryoelectron Microscopy , Protein Binding , Protein Structure, TertiaryABSTRACT
The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.