Nature Structural & Molecular Biology

Research highlights.

Publication Date: 2008 May PMID: 18461046
Authors:
Journal: Nat Struct Mol Biol

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New clues to actin function in chromatin regulation.

Publication Date: 2008 May PMID: 18461045
Authors: Gottschalk, A. J. - Conaway, R. C. - Conaway, J. W.
Journal: Nat Struct Mol Biol

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DNA damage: a histone-code mediator leaves the stage.

Publication Date: 2008 May PMID: 18461044
Authors: Lukas, J. - Bartek, J.
Journal: Nat Struct Mol Biol

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The type II secretion arrowhead: the structure of GspI-GspJ-GspK.

Publication Date: 2008 May PMID: 18461043
Authors: Forest, K. T.
Journal: Nat Struct Mol Biol

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Modifications target spliceosome dynamics.

Publication Date: 2008 May PMID: 18461042
Authors: Maeder, C. - Guthrie, C.
Journal: Nat Struct Mol Biol

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Promoting creativity and innovation.

Publication Date: 2008 May PMID: 18461041
Authors:
Journal: Nat Struct Mol Biol

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Membrane-dependent signal integration by the Ras activator Son of sevenless.

Publication Date: 2008 May PMID: 18454158
Authors: Gureasko, J. - Galush, W. J. - Boykevisch, S. - Sondermann, H. - Bar-Sagi, D. - Groves, J. T. - Kuriyan, J.
Journal: Nat Struct Mol Biol

The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOS(cat)) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOS(cat) to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition.

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The structural basis for cap binding by influenza virus polymerase subunit PB2.

Publication Date: 2008 May PMID: 18454157
Authors: Guilligay, D. - Tarendeau, F. - Resa-Infante, P. - Coloma, R. - Crepin, T. - Sehr, P. - Lewis, J. - Ruigrok, R. W. - Ortin, J. - Hart, D. J. - Cusack, S.
Journal: Nat Struct Mol Biol

Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.

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The exonuclease ERI-1 has a conserved dual role in 5.8S rRNA processing and RNAi.

Publication Date: 2008 May PMID: 18438419
Authors: Gabel, H. W. - Ruvkun, G.
Journal: Nat Struct Mol Biol

The exonuclease ERI-1 negatively regulates RNA interference in Caenorhabditis elegans and Schizosaccharomyces pombe, and is required for production of some C. elegans endogenous small interfering RNAs. We show that ERI-1 performs 3' end processing of the 5.8S ribosomal RNA in both C. elegans and S. pombe. In C. elegans, two protein isoforms of ERI-1 are localized to the cytoplasm, and each has distinct functions in ribosomal RNA processing and negative regulation of RNA interference.

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Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA processing.

Publication Date: 2008 May PMID: 18438418
Authors: Ansel, K. M. - Pastor, W. A. - Rath, N. - Lapan, A. D. - Glasmacher, E. - Wolf, C. - Smith, L. C. - Papadopoulou, N. - Lamperti, E. D. - Tahiliani, M. - Ellwart, J. W. - Shi, Y. - Kremmer, E. - Rao, A. - Heissmeyer, V.
Journal: Nat Struct Mol Biol

Eri1 is a 3'-to-5' exoribonuclease conserved from fission yeast to humans. Here we show that Eri1 associates with ribosomes and ribosomal RNA (rRNA). Ribosomes from Eri1-deficient mice contain 5.8S rRNA that is aberrantly extended at its 3' end, and Eri1, but not a catalytically inactive mutant, converts this abnormal 5.8S rRNA to the wild-type form in vitro and in cells. In human and murine cells, Eri1 localizes to the cytoplasm and nucleus, with enrichment in the nucleolus, the site of preribosome biogenesis. RNA binding residues in the Eri1 SAP and linker domains promote stable association with rRNA and thereby facilitate 5.8S rRNA 3' end processing. Taken together, our findings indicate that Eri1 catalyzes the final trimming step in 5.8S rRNA processing, functionally and spatially connecting this regulator of RNAi with the basal translation machinery.

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Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system.

Publication Date: 2008 May PMID: 18438417
Authors: Korotkov, K. V. - Hol, W. G.
Journal: Nat Struct Mol Biol

Gram-negative bacteria translocate various proteins including virulence factors across their outer membrane via type 2 secretion systems (T2SSs). T2SSs are thought to contain a pseudopilus, a subcomplex formed by one major and several minor pseudopilins. We report the crystal structure of the complex formed by three minor pseudopilins from enterotoxigenic Escherichia coli. The GspK-GspI-GspJ complex has quasihelical characteristics and an architecture consistent with a localization at the pseudopilus tip. The alpha-domain of GspK has a previously unobserved fold with an unexpected dinuclear metal binding site. The area surrounding its disulfide bridge is conserved and might interact with other T2SS components or with secreted proteins.

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A role for ubiquitin in the spliceosome assembly pathway.

Publication Date: 2008 May PMID: 18425143
Authors: Bellare, P. - Small, E. C. - Huang, X. - Wohlschlegel, J. A. - Staley, J. P. - Sontheimer, E. J.
Journal: Nat Struct Mol Biol

The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitin's protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.

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Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome.

Publication Date: 2008 May PMID: 18425142
Authors: Mathew, R. - Hartmuth, K. - Mohlmann, S. - Urlaub, H. - Ficner, R. - Luhrmann, R.
Journal: Nat Struct Mol Biol

Several protein kinases, including SRPK1 and SRPK2, have been implicated in spliceosome assembly and catalytic activation. However, little is known about their targets. Here we show that SRPK1 is predominantly associated with U1 small nuclear ribonucleoprotein (snRNP), whereas SRPK2 associates with the U4/U6-U5 tri-snRNP. RNAi-mediated depletion in HeLa cells showed that SRPK2 is essential for cell viability, and it is required for spliceosomal B complex formation. SRPK2 knock down results in hypophosphorylation of the arginine-serine (RS) domain-containing human PRP28 protein (PRP28, also known as DDX23), and destabilizes PRP28 association with the tri-snRNP. Immunodepletion of PRP28 from HeLa cell nuclear extract and complementation studies revealed that PRP28 phosphorylation is required for its stable association with the tri-snRNP and for tri-snRNP integration into the B complex. Our results demonstrate a role for SRPK2 in splicing and reveal a previously unknown function for PRP28 in spliceosome assembly.

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Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNA(Cys).

Publication Date: 2008 May PMID: 18425141
Authors: Zhang, C. M. - Liu, C. - Slater, S. - Hou, Y. M.
Journal: Nat Struct Mol Biol

Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys).

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The HSA domain binds nuclear actin-related proteins to regulate chromatin-remodeling ATPases.

Publication Date: 2008 May PMID: 18408732
Authors: Szerlong, H. - Hinata, K. - Viswanathan, R. - Erdjument-Bromage, H. - Tempst, P. - Cairns, B. R.
Journal: Nat Struct Mol Biol

We identify the helicase-SANT-associated (HSA) domain as the primary binding platform for nuclear actin-related proteins (ARPs) and actin. Individual HSA domains from chromatin remodelers (RSC, yeast SWI-SNF, human SWI-SNF, SWR1 and INO80) or modifiers (NuA4) reconstitute their respective ARP-ARP or ARP-actin modules. In RSC, the HSA domain resides on the catalytic ATPase subunit Sth1. The Sth1 HSA is essential in vivo, and its omission causes the specific loss of ARPs and a moderate reduction in ATPase activity. Genetic selections for arp suppressors yielded specific gain-of-function mutations in two new domains in Sth1, the post-HSA domain and protrusion 1, which are essential for RSC function in vivo but not ARP association. Taken together, we define the role of the HSA domain and provide evidence for a regulatory relationship involving the ARP-HSA module and two new functional domains conserved in remodeler ATPases that contain ARPs.

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DNA apurinic-apyrimidinic site binding and excision by endonuclease IV.

Publication Date: 2008 May PMID: 18408731
Authors: Garcin, E. D. - Hosfield, D. J. - Desai, S. A. - Haas, B. J. - Bjoras, M. - Cunningham, R. P. - Tainer, J. A.
Journal: Nat Struct Mol Biol

Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three-metal ion mechanism for damage binding and incision. The 1.10-A resolution DNA-free and the 2.45-A resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-A resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV alpha-beta triose phosphate isomerase (TIM) barrel and APE1 four-layer alpha-beta folds of the apurinic-apyrimidinic endonuclease families.

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ATP-dependent chromatin remodeling shapes the DNA replication landscape.

Publication Date: 2008 May PMID: 18408730
Authors: Vincent, J. A. - Kwong, T. J. - Tsukiyama, T.
Journal: Nat Struct Mol Biol

The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 have especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes that promote DNA replication and define a specific stage of replication that requires remodeling for normal function.

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Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel.

Publication Date: 2008 May PMID: 18391966
Authors: Bornemann, T. - Jockel, J. - Rodnina, M. V. - Wintermeyer, W.
Journal: Nat Struct Mol Biol

Ribosomes synthesizing inner membrane proteins in Escherichia coli are targeted to the translocon in the plasma membrane by the signal recognition particle (SRP) and the SRP receptor, FtsY. Here we show using a purified system that membrane targeting does not require an exposed signal-anchor sequence, as SRP-dependent targeting takes place with ribosomes containing short nascent peptides, with or without a signal-anchor sequence, within the peptide exit tunnel. Signaling from inside the tunnel involves ribosomal protein L23, which constitutes part of the SRP binding site. When nascent peptides emerge from the ribosome, the targeting complex is maintained with ribosomes exposing a signal-anchor sequence, whereas ribosomes exposing other sequences are released. These results indicate that ribosome-nascent chain complexes containing any nascent peptide within the exit tunnel can enter the SRP targeting pathway to be sorted at the membrane into ribosome-nascent chain complexes that synthesize either membrane or cytosolic proteins.

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A conserved rRNA methyltransferase regulates ribosome biogenesis.

Publication Date: 2008 May PMID: 18391965
Authors: Xu, Z. - O'Farrell, H. C. - Rife, J. P. - Culver, G. M.
Journal: Nat Struct Mol Biol

In contrast to the diversity of most ribosomal RNA modification patterns and systems, the KsgA methyltransferase family seems to be nearly universally conserved along with the modifications it catalyzes. Our data reveal that KsgA interacts with small ribosomal subunits near functional sites, including Initiation factor 3 and 50S subunit binding sites. These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation.

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Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis.

Publication Date: 2008 May PMID: 18391964
Authors: Ptacin, J. L. - Nollmann, M. - Becker, E. C. - Cozzarelli, N. R. - Pogliano, K. - Bustamante, C.
Journal: Nat Struct Mol Biol

In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to study the properties of DNA translocation by SpoIIIE in vitro and in vivo. These data allow us to propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.

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