Molecular Cell

Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging.

Publication Date: 2008 May 9 PMID: 18471983
Authors: Park, W. S. - Heo, W. D. - Whalen, J. H. - O'Rourke, N. A. - Bryan, H. M. - Meyer, T. - Teruel, M. N.
Journal: Mol Cell

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.

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To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.

Publication Date: 2008 May 9 PMID: 18471982
Authors: Dewson, G. - Kratina, T. - Sim, H. W. - Puthalakath, H. - Adams, J. M. - Colman, P. M. - Kluck, R. M.
Journal: Mol Cell

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.

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Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases.

Publication Date: 2008 May 9 PMID: 18471981
Authors: Rabl, J. - Smith, D. M. - Yu, Y. - Chang, S. C. - Goldberg, A. L. - Cheng, Y.
Journal: Mol Cell

Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring alpha subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the alpha subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause alpha subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.

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Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation.

Publication Date: 2008 May 9 PMID: 18471980
Authors: Fei, J. - Kosuri, P. - MacDougall, D. D. - Gonzalez, R. L. Jr
Journal: Mol Cell

By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.

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Regulation of estrogen receptor alpha by the SET7 lysine methyltransferase.

Publication Date: 2008 May 9 PMID: 18471979
Authors: Subramanian, K. - Jia, D. - Kapoor-Vazirani, P. - Powell, D. R. - Collins, R. E. - Sharma, D. - Peng, J. - Cheng, X. - Vertino, P. M.
Journal: Mol Cell

Estrogen receptor alpha (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate "ER modification cassettes" that yield distinct patterns for signaling downstream events.

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Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates.

Publication Date: 2008 May 9 PMID: 18471978
Authors: Li, F. - Dong, J. - Pan, X. - Oum, J. H. - Boeke, J. D. - Lee, S. E.
Journal: Mol Cell

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.

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Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation.

Publication Date: 2008 May 9 PMID: 18471977
Authors: Batra, V. K. - Beard, W. A. - Shock, D. D. - Pedersen, L. C. - Wilson, S. H.
Journal: Mol Cell

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.

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The metastasis-associated gene Prl-3 Is a p53 target involved in cell-cycle regulation.

Publication Date: 2008 May 9 PMID: 18471976
Authors: Basak, S. - Jacobs, S. B. - Krieg, A. J. - Pathak, N. - Zeng, Q. - Kaldis, P. - Giaccia, A. J. - Attardi, L. D.
Journal: Mol Cell

The p53 tumor suppressor restricts tumorigenesis through the transcriptional activation of target genes involved in cell-cycle arrest and apoptosis. Here, we identify Prl-3 (phosphatase of regenerating liver-3) as a p53-inducible gene. Whereas previous studies implicated Prl-3 in metastasis because of its overexpression in metastatic human colorectal cancer and its ability to promote invasiveness and motility, we demonstrate here that Prl-3 is an important cell-cycle regulator. Consistent with a role in DNA damage-induced cell-cycle arrest, Prl-3 overexpression induces G(1) arrest downstream of p53 by triggering a PI3K-Akt-activated negative feedback loop. Surprisingly, attenuation of Prl-3 expression also elicits an arrest response, suggesting that basal level Prl-3 expression is pivotal for normal cell-cycle progression. Our findings highlight key dose-dependent functions of Prl-3 in both positive and negative regulation of cell-cycle progression and provide insight into Prl-3's role in cancer progression.

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Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A.

Publication Date: 2008 May 9 PMID: 18471975
Authors: Wolthuis, R. - Clay-Farrace, L. - van Zon, W. - Yekezare, M. - Koop, L. - Ogink, J. - Medema, R. - Pines, J.
Journal: Mol Cell

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.

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Recurrent initiation: a mechanism for triggering p53 pulses in response to DNA damage.

Publication Date: 2008 May 9 PMID: 18471974
Authors: Batchelor, E. - Mock, C. S. - Bhan, I. - Loewer, A. - Lahav, G.
Journal: Mol Cell

DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53's dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM, which is reactivated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage.

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Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage.

Publication Date: 2008 May 9 PMID: 18471973
Authors: Bonilla, C. Y. - Melo, J. A. - Toczyski, D. P.
Journal: Mol Cell

Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.

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AMPK and Raptor: matching cell growth to energy supply.

Publication Date: 2008 May 9 PMID: 18471972
Authors: Hardie, D. G.
Journal: Mol Cell

In a recent issue of Molecular Cell, Gwinn et al. (2008) suggest a novel mechanism by which AMPK signals to mTOR, and they provide new insight into how information about cellular energy status is fed into decisions about cell growth and proliferation.

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SF2/ASF TORCs up translation.

Publication Date: 2008 May 9 PMID: 18471971
Authors: Bushell, M. - Stoneley, M. - Spriggs, K. A. - Willis, A. E.
Journal: Mol Cell

In a recent issue of Molecular Cell, Michlewski et al. (2008) show that SF2/ASF, a splicing factor, stimulates translation initiation by directly recruiting the mammalian target of rapamycin (mTOR) to a subset of mRNAs.

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Too much of a good thing: the Prl-3 in p53's oyster.

Publication Date: 2008 May 9 PMID: 18471970
Authors: Hinds, P. W.
Journal: Mol Cell

In this issue of Molecular Cell, Basak et al. (2008) identify Prl-3 as a p53-inducible gene acting in DNA damage-induced cell-cycle arrest. Both deletion and overexpression of Prl-3 can be cytostatic, adding to a growing list of genes whose expression must be carefully titrated for proper cell proliferation.

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DNA polymerases at the replication fork in eukaryotes.

Publication Date: 2008 May 9 PMID: 18471969
Authors: Stillman, B.
Journal: Mol Cell

The Kunkel laboratory has recently assigned polymerase (Pol) epsilon as the leading strand polymerase. In a recent issue of Molecular Cell, they now assign Pol delta as the lagging strand polymerase.

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DAP5 Promotes Cap-Independent Translation of Bcl-2 and CDK1 to Facilitate Cell Survival during Mitosis.

Publication Date: 2008 Apr 30 PMID: 18450493
Authors: Marash, L. - Liberman, N. - Henis-Korenblit, S. - Sivan, G. - Reem, E. - Elroy-Stein, O. - Kimchi, A.
Journal: Mol Cell

DAP5 is an eIF4G protein previously implicated in mediating cap-independent translation in response to cellular stresses. Here we report that DAP5 is crucial for continuous cell survival in nonstressed cells. The knockdown of endogenous DAP5 induced M phase-specific caspase-dependent apoptosis. Bcl-2 and CDK1 were identified by two independent screens as DAP5 translation targets. Notably, the activity of the Bcl-2 IRES was reduced in DAP5 knockdown cells and a selective shift of Bcl-2 mRNA toward light polysomal fractions was detected. Furthermore, a functional IRES was identified in the 5'UTR of CDK1. At the cellular level, attenuated translation of CDK1 by DAP5 knockdown decreased the phosphorylation of its M phase substrates. Ectopic expression of Bcl-2 or CDK1 proteins partially reduced the extent of caspase activation caused by DAP5 knockdown. Thus, DAP5 is necessary for maintaining cell survival during mitosis by promoting cap-independent translation of at least two prosurvival proteins.

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Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae.

Publication Date: 2008 Apr 25 PMID: 18439903
Authors: Ben-Aroya, S. - Coombes, C. - Kwok, T. - O'Donnell, K. A. - Boeke, J. D. - Hieter, P.
Journal: Mol Cell

The Saccharomyces cerevisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.

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PHAPI, CAS, and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1.

Publication Date: 2008 Apr 25 PMID: 18439902
Authors: Kim, H. E. - Jiang, X. - Du, F. - Wang, X.
Journal: Mol Cell

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.

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GGA and Arf proteins modulate retrovirus assembly and release.

Publication Date: 2008 Apr 25 PMID: 18439901
Authors: Joshi, A. - Garg, H. - Nagashima, K. - Bonifacino, J. S. - Freed, E. O.
Journal: Mol Cell

The Gag protein is the major structural determinant of retrovirus assembly. Although a number of cellular factors have been reported to facilitate retrovirus release, little is known about the cellular machinery that directs Gag to the site of virus assembly. Here, we report roles for the Golgi-localized gamma-ear containing Arf-binding (GGA) and ADP ribosylation factor (Arf) proteins in retrovirus particle assembly and release. Whereas siRNA-mediated depletion of GGA2 and GGA3 led to a significant increase in particle release in a late domain-dependent manner, GGA overexpression severely reduced retrovirus particle production by impairing Gag trafficking to the membrane. GGA overexpression inhibited retroviral assembly and release by disrupting Arf protein activity. Furthermore, disruption of endogenous Arf activity inhibited particle production by decreasing Gag-membrane binding. These findings identify the GGA proteins as modulators of HIV-1 release and the Arf proteins as critical cellular cofactors in retroviral Gag trafficking to the plasma membrane.

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AMPK phosphorylation of raptor mediates a metabolic checkpoint.

Publication Date: 2008 Apr 25 PMID: 18439900
Authors: Gwinn, D. M. - Shackelford, D. B. - Egan, D. F. - Mihaylova, M. M. - Mery, A. - Vasquez, D. S. - Turk, B. E. - Shaw, R. J.
Journal: Mol Cell

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.

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PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival.

Publication Date: 2008 Apr 25 PMID: 18439899
Authors: Bozulic, L. - Surucu, B. - Hynx, D. - Hemmings, B. A.
Journal: Mol Cell

Protein kinase B (PKB/Akt) is a well-established regulator of several essential cellular processes. Here, we report a route by which activated PKB promotes survival in response to DNA insults in vivo. PKB activation following DNA damage requires 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localizes in the nucleus of gamma-irradiated cells adjacent to DNA double-strand breaks, where it colocalizes and interacts with DNA-PK. Levels of active PKB inversely correlate with DNA damage-induced apoptosis. A significant portion of p53- and DNA damage-regulated genes are misregulated in cells lacking PKBalpha. PKBalpha knockout mice show impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single-dose whole-body irradiation. Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a prosurvival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKBalpha isoform.

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Molecular mechanism of drug-dependent ribosome stalling.

Publication Date: 2008 Apr 25 PMID: 18439898
Authors: Vazquez-Laslop, N. - Thum, C. - Mankin, A. S.
Journal: Mol Cell

Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.

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The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1.

Publication Date: 2008 Apr 25 PMID: 18439897
Authors: Michlewski, G. - Sanford, J. R. - Caceres, J. F.
Journal: Mol Cell

The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.

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Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.

Publication Date: 2008 Apr 25 PMID: 18439896
Authors: Witte, G. - Hartung, S. - Buttner, K. - Hopfner, K. P.
Journal: Mol Cell

To reveal mechanisms of DNA damage checkpoint initiation, we structurally and biochemically analyzed DisA, a protein that controls a Bacillus subtilis sporulation checkpoint in response to DNA double-strand breaks. We find that DisA forms a large octamer that consists of an array of an uncharacterized type of nucleotide-binding domain along with two DNA-binding regions related to the Holliday junction recognition protein RuvA. Remarkably, the nucleotide-binding domains possess diadenylate cyclase activity. The resulting cyclic diadenosine phosphate, c-di-AMP, is reminiscent but distinct from c-di-GMP, an emerging prokaryotic regulator of complex cellular processes. Diadenylate cyclase activity is unaffected by linear DNA or DNA ends but strongly suppressed by branched nucleic acids such as Holliday junctions. Our data indicate that DisA signals DNA structures that interfere with chromosome segregation via c-di-AMP. Identification of the diadenylate cyclase domain in other eubacterial and archaeal proteins implies a more general role for c-di-AMP in prokaryotes.

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An ARS element inhibits DNA replication through a SIR2-dependent mechanism.

Publication Date: 2008 Apr 25 PMID: 18439895
Authors: Crampton, A. - Chang, F. - Pappas, D. L. Jr - Frisch, R. L. - Weinreich, M.
Journal: Mol Cell

During G1 phase, a prereplicative complex (pre-RC) that determines where DNA synthesis initiates forms at origins. The Sir2p histone deacetylase inhibits pre-RC assembly at a subset of origins, suggesting that Sir2p inhibits DNA replication through a unique aspect of origin structure. Here, we identified five SIR2-sensitive origins on chromosomes III and VI. Linker scan analysis of two origins indicated that they share a common organization, including an inhibitory sequence positioned 3' to the sites of origin recognition complex (ORC) binding and pre-RC assembly. This inhibitory sequence (I(S)) required SIR2 for its activity, suggesting that SIR2 inhibits origins through this sequence. Furthermore, I(S) elements occurred within positioned nucleosomes, and Abf1p-mediated exclusion of nucleosomes from the origin abrogated the inhibition. These data suggest that Sir2p and I(S) elements inhibit origin activity by promoting an unfavorable chromatin structure for pre-RC assembly.

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Architecture of a serine recombinase-DNA regulatory complex.

Publication Date: 2008 Apr 25 PMID: 18439894
Authors: Mouw, K. W. - Rowland, S. J. - Gajjar, M. M. - Boocock, M. R. - Stark, W. M. - Rice, P. A.
Journal: Mol Cell

An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.

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Division of labor at the eukaryotic replication fork.

Publication Date: 2008 Apr 25 PMID: 18439893
Authors: Nick McElhinny, S. A. - Gordenin, D. A. - Stith, C. M. - Burgers, P. M. - Kunkel, T. A.
Journal: Mol Cell

DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many years. Here we investigate the contribution of Pol delta to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol delta allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T x dGTP) than for its complement (A x dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol delta replication is performed using the lagging strand template. When combined with recent evidence implicating Pol epsilon in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol epsilon and Pol delta, respectively.

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IAPs: what's in a name?

Publication Date: 2008 Apr 25 PMID: 18439892
Authors: Srinivasula, S. M. - Ashwell, J. D.
Journal: Mol Cell

Originally described in insect viruses, cellular proteins with Baculoviral IAP repeat (BIR) motifs have been thought to function primarily as inhibitors of apoptosis. The subsequent finding that a subset of IAPs that contain a RING domain have ubiquitin protein ligase (E3) activity implied the presence of other functions. It is now known that IAPs are involved in mitotic chromosome segregation, cellular morphogenesis, copper homeostasis, and intracellular signaling. Here, we review the current understanding of the roles of IAPs in apoptotic and nonapoptotic processes and explore the notion that the latter represents the primary physiologic activities of IAPs.

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A Three-Dimensional Model of a Group II Intron RNA and Its Interaction with the Intron-Encoded Reverse Transcriptase.

Publication Date: 2008 Apr 16 PMID: 18424209
Authors: Dai, L. - Chai, D. - Gu, S. Q. - Gabel, J. - Noskov, S. Y. - Blocker, F. J. - Lambowitz, A. M. - Zimmerly, S.
Journal: Mol Cell

Group II introns are self-splicing ribozymes believed to be the ancestors of spliceosomal introns. Many group II introns encode reverse transcriptases that promote both RNA splicing and intron mobility to new genomic sites. Here we used a circular permutation and crosslinking method to establish 16 intramolecular distance relationships within the mobile Lactococcus lactis Ll.LtrB-DeltaORF intron. Using these new constraints together with 13 established tertiary interactions and eight published crosslinks, we modeled a complete three-dimensional structure of the intron. We also used the circular permutation strategy to map RNA-protein interaction sites through fluorescence quenching and crosslinking assays. Our model provides a comprehensive structural framework for understanding the function of group II ribozymes, their natural structural variations, and the mechanisms by which the intron-encoded protein promotes RNA splicing and intron mobility. The model also suggests an arrangement of active site elements that may be conserved in the spliceosome.

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