Content
2009
Modulation of protein properties in living cells using nanobodies
13.12.2009
Nature Structural & Molecular Biology,
2009,
doi:10.1038/nsmb.1727
published on 13.12.2009
Nature Structural & Molecular Biology, online article
Nature Structural & Molecular Biology, online article
Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP–nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.
PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)
04.12.2009
Plastid-specific ribosomal proteins (PSRPs) have been proposed
to play roles in the light-dependent regulation of chloroplast
translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal
that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation.Wefind that the presence ofPSRP1/pYlowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.
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Structural Insight into Nascent Polypeptide Chain–Mediated Translational Stalling
04.12.2009
Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom–resolution cryo–electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.
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Structure of Monomeric Yeast and Mammalian Sec61 Complexes Interacting with the Translating Ribosome
04.12.2009
The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo–electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.
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The A–Z of bacterial translation inhibitors
01.12.2009
Crit. Rev. Biochem. Mol.,
2009,
44(6),
393-433, doi:10.3109/10409230903307311
published on 01.12.2009
Crit. Rev. Biochem. Mol., online article
Crit. Rev. Biochem. Mol., online article
Protein synthesis is one of the major targets in the cell for antibiotics. This review endeavors to provide a comprehensive “post-ribosome structure” A–Z of the huge diversity of antibiotics that target the bacterial translation apparatus, with an emphasis on correlating the vast wealth of biochemical data with more recently available ribosome structures, in order to understand function. The binding site, mechanism of action, and modes of resistance for 26 different classes of protein synthesis inhibitors are presented, ranging from ABT-773 to Zyvox. In addition to improving our understanding of the process of translation, insight into the mechanism of action of antibiotics is essential to the development of novel and more effective antimicrobial agents to combat emerging bacterial resistance to many clinically-relevant drugs.
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Biosynthesis of the Aminocyclitol Subunit of Hygromycin A in Streptomyces hygroscopicus NRRL 2388
25.11.2009
Chem. Biol.,
2009,
16(11),
1180-1189, doi:10.1016/j.chembiol.2009.10.013
published on 25.11.2009
Chem. Biol., online article
Chem. Biol., online article
The antibacterial activity of hygromycin A (HA) arises from protein synthesis inhibition and is dependent upon a methylenedioxy bridged-aminocyclitol moiety. Selective gene deletions and chemical complementation in Streptomyces hygroscopicus NRRL 2388 showed that the hyg18 and hyg25 gene products, proposed to generate a myo-inositol intermediate, are dispensable for HA biosynthesis but contribute to antibiotic yields. Hyg8 and Hyg17, proposed to introduce the amine functionality, are essential for HA biosynthesis. Hyg6 is a methyltransferase acting on the aminocyclitol, and a Δhyg6 mutant produces desmethylenehygromycin A. Deletion of hyg7, a metallo-dependant hydrolase homolog gene, resulted in methoxyhygromycin A production, demonstrating that the corresponding gene product is responsible for the proposed oxidative cyclization step of methylenedioxy bridge formation. The methyl/methylene group is not required for in vitro protein synthesis inhibition but is essential for activity against Escherichia coli.
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Identification of Distinct Thiopeptide-Antibiotic Precursor Lead Compounds Using Translation Machinery Assays
30.10.2009
Chem. Biol.,
2009,
16(10),
1087-1096, doi:10.1016/j.chembiol.2009.09.016
published on 30.10.2009
Chem. Biol., online article
Chem. Biol., online article
Most thiopeptide antibiotics target the translational machinery: thiostrepton (ThS) and nosiheptide (NoS) target the ribosome and inhibit translation factor function, whereas GE2270A/T binds to the elongation factor EF-Tu and prevents ternary complex formation. We have used several in vitro translational machinery assays to screen a library of thiopeptide antibiotic precursor compounds and identified four families of precursor compounds that are either themselves inhibitory or are able to relieve the inhibitory effects of ThS, NoS, or GE2270T. Some of these precursors represent distinct compounds with respect to their ability to bind to ribosomes. The results not only provide insight into the mechanism of action of thiopeptide compounds but also demonstrate the potential of such assays for identifying lead compounds that might be missed using conventional inhibitory screening protocols.
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X-ray structure of Pur-α reveals a Whirly-like fold and an unusual nucleic-acid binding surface
21.10.2009
The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA- and DNA-binding. Its best-studied family member, Pur-α, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-α-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-α is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-α, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed “PUR repeats,” which interact with each other to form a PUR domain. DNA- and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave β-sheets. Structure-based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size- exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-α. Surface envelopes calculated from SAXS data show that the Pur-α dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-α has direct implications for ssDNA/ssRNA and dsDNA binding.
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Lessons from structural and biochemical studies on the archaeal exosome
10.10.2009
Biochem. Soc. Trans.,
2009,
37,
83-87, doi:10.1042/BST0370083
published on 10.10.2009
Biochem. Soc. Trans., online article
Biochem. Soc. Trans., online article
The RNA exosome is a multisubunit exonuclease involved in numerous RNA maturation and degradation processes. Exosomes are found in eukaryotes and archaea and are related to bacterial polynucleotide phosphorylates. Over the past years structural and biochemical analysis revealed that archaeal exosomes have a large processing chamber with three phosphorolytic active sites that degrade RNA in the 3′→5′ direction in a highly processive manner. A narrow entry pore, framed by putative RNA-binding domains, could account for the high processivity and also prevent degradation of structured RNA. The phosphorolytic nuclease activity is reversible, leading to formation of heteropolymeric tails from nucleoside diphosphates as substrate. This reversibility is difficult to regulate, suggesting why, during evolution and emergence of stable poly(A) tails in eukaryotes, polyadenylation and nuclease activities in the human exosome and associated factors have been separated.
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Enhanced SnapShot: Antibiotic Inhibition of Protein Synthesis II
02.10.2009
Cell,
2009,
139(1),
pp. 212 - 212.e1, doi:10.1016/j.cell.2009.08.009
published on 02.10.2009
Cell, online article
Cell, online article
The translational apparatus is one of the major targets for antibiotics in the bacterial cell. Antibiotics predominantly interact with the functional centers of the ribosome, namely the messenger RNA (mRNA)-transfer RNA (tRNA) decoding region on the 30S subunit, the peptidyltransferase center on the 50S subunit, or the ribosomal exit tunnel through which the nascent polypeptide chain passes during translation. Protein synthesis can be divided into three phases: initiation, elongation, and termination/recycling.
DNA Double-Strand Breaks Come into Focus
02.10.2009
The Mre11-Rad50-Nbs1 (MRN) complex senses DNA double-strand breaks and recruits different repair pathway and checkpoint proteins to break foci. Two new studies (Williams et al., 2009; Lloyd et al., 2009,Williams et al., 2009; Lloyd et al., 2009) identify Nbs1 as a key factor in this process and reveal how an N-terminal protein recruitment module in Nbs1 binds to different response factors through shared phosphopeptide motifs.
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SnapShot: Antibiotic Inhibition of Protein Synthesis I
18.09.2009
Cell,
2009,
138,
1248-1248e1, doi:10.1016/j.cell.2009.08.001
published on 18.09.2009
Cell, online article
Cell, online article
The translational apparatus is one of the major targets for antibiotics in the bacterial cell. Antibiotics predominantly interact with the functional centers of the ribosome, namely the messenger RNA (mRNA)-transfer RNA (tRNA) decoding region on the 30S subunit, the peptidyltransferase center on the 50S subunit, or the ribosomal exit tunnel through which the nascent polypeptide chain passes during translation. Protein synthesis can be divided into three phases: initiation, elongation, and termination/recycling.
The torpedo nuclease Rat1 is insufficient to terminate RNA polymerase II in vitro
17.09.2009
J. Biol. Chem.,
2009,
in press, doi:10.1074/jbc.M109.013847
published on 17.09.2009
J. Biol. Chem., online article
J. Biol. Chem., online article
Termination of RNA polymerase (Pol) II transcription in vivo requires the 5’-RNA exonuclease Rat1. It was proposed that Rat1 degrades RNA from the 5’-end that is created by transcript cleavage, catches up with elongating Pol II, and acts like a torpedo that removes Pol II from DNA. Here we test the torpedo model in an in vitro system based on bead-coupled Pol II elongation complexes (ECs). Recombinant Rat1 complexes with Rai1, and with Rai1 and Rtt103, degrade RNA extending from the EC until they reach the polymerase surface, but fail to terminate Pol II. Instead, the EC retains an ~18 nt RNA that remains with its 3’-end at the active site and can be elongated. Thus, Pol II termination apparently requires a factor or several factors in addition to Rat1, Rai1 and Rtt103, post-translational modifications of these factors, or unusual reaction conditions.
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Molecular basis of transcriptional mutagenesis at 8-oxoguanine
16.09.2009
J. Biol. Chem.,
2009,
284,
31658-31663, doi:10.1074/jbc.M109.022764
published on 16.09.2009
J. Biol. Chem., online article
J. Biol. Chem., online article
Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site. The misincorporated adenine escapes intrinsic proofreading, resulting in transcriptional mutagenesis that is observed directly by mass spectrometric RNA analysis.
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The Final Step of Hygromycin A Biosynthesis, Oxidation of C-5''-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux
12.09.2009
Antimicrob. Agents Ch.,
2009,
53(12),
5163-5172, doi:10.1128/AAC.01069-09
published on 12.09.2009
Antimicrobial Agents and Chemotherapy, online article
Antimicrobial Agents and Chemotherapy, online article
Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5''-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in Escherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the {Delta}hyg19 or the {Delta}hyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.
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Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization
26.08.2009
In eukaryotic cells, dozens to hundreds of different mRNAs are localized by specialized motor-dependent transport complexes. One of the best-studied examples for directional mRNA transport is the localization of ASH1 mRNA in Saccharomyces cerevisiae. For transport, ASH1 mRNA is bound by the unusual RNA-binding protein She2p. Although previous results indicated that She2p forms dimers required for RNA binding and transcript localization, it remained unclear if the dimer constitutes the minimal RNA-binding unit assembling in vivo. By using analytical ultracentrifugation we found that She2p forms larger oligomeric complexes in solution. We also identified a point mutant that shows impaired oligomer formation. Size-exclusion chromatography suggests that She2p forms defined tetramers at physiological concentrations. Subsequent structural studies by small-angle X-ray scattering confirmed this finding and demonstrated that the previously observed She2p dimers interact in a head-to-head conformation to form an elongated tetrameric complex. This She2p tetramer suggests the generation of large continuous RNA-binding surfaces at both sides of the complex. Biochemical studies and immunostaining of cells confirmed that She2p tetramer formation is required for RNA binding, efficient mRNP assembly, and mRNA localization in vivo. Our finding on She2p tetramerization resolves previously raised questions on complex formation and mRNP function.
Hsp90 is regulated by a switch point in the C-terminal domain
21.08.2009
EMBO reports,
2009,
1147 - 1153,
doi:10.1038/embor.2009.153
published on 21.08.2009
EMBO reports, online article
EMBO reports, online article
Heat shock protein 90 (Hsp90) is an abundant, dimeric ATPdependent molecular chaperone, and ATPase activity is essential for its in vivo functions. S-nitrosylation of a residue located in the carboxy-terminal domain has been shown to affect Hsp90 activity in vivo. To understand how variation of a specific amino acid far away from the amino-terminal ATP-binding site regulates Hsp90 functions, we mutated the corresponding residue and analysed yeast and human Hsp90 variants both in vivo and in vitro. Here, we show that this residue is a conserved, strong regulator of Hsp90 functions, including ATP hydrolysis and chaperone activity. Unexpectedly, the variants alter both the C-terminal and N-terminal association properties of Hsp90, and shift its conformational equilibrium within the ATPase cycle. Thus,
S-nitrosylation of this residue allows the fast and efficient fine
regulation of Hsp90.
Prolonged stability by cyclization: Macrocyclic phosphino dipeptide isostere inhibitors of b-secretase (BACE1)
01.08.2009
Bioorganic & Medicinal Chemistry Letters,
2009,
19,
4427 - 4431
published on 01.08.2009
Cyclization of recently reported linear phosphino dipeptide isostere inhibitors of BACE1 via side chain olefin metathesis yielded macrocyclic BACE1 inhibitors. The most potent compound II-P1 (IC50 of 47 nM) and the corresponding linear analog I were tested for serum stability. The approach led to three times prolonged half life serum stability of 44 min for the macrocyclic inhibitor II-P1 compared to the linear compound I.
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Endo-siRNAs depend on a new isoform of loquacious and target artificially introduced, high-copy sequences
04.08.2009
Transposons are mobile genetic elements found in the hereditary material of humans and other organisms. They can replicate and the new copies can insert at novel sites in the genome. Because this threatens the whole organism, molecular mechanisms have evolved which can repress transposon activity. CIPSM-Professor Klaus Förstemann of the Gene Center of Ludwig-Maximilians-Universität (LMU) in Munich and a team of researchers working with the fruitfly Drosophila melanogaster have now uncovered a new type of cellular defence that acts against DNA sequences present in high copy numbers inside the cell, even if they have not integrated into the genome. Small molecules of RNA (a class of nucleic acid closely related to the genetic material DNA) play the central role. “Transposons are genomic parasites, so to speak”, says Förstemann. “If they are allowed to proliferate, the genome can become unstable or cancers can develop. We now want to find out whether mammalian cells possess this newly discovered defence mechanism and to elucidate precisely how it works.
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Press
Transposons constitute a significant fraction of the genomes of most higher organisms. Indeed, it is estimated that these mobile elements, which include one or more genes, make up as much as half of the genetic material. “This demonstrates”, says Förstemann, “that it is not always possible to tame these “selfish” genetic elements, although highly efficient mechanisms of defence have evolved. For instance, in the germ cells, which are required for reproduction, the system of so-called piRNAs ensures that transposon activity is inhibited – but only if these RNAs are transmitted from the mother. Disruption of this system usually leads to a drastic reduction in the fertility of the progeny.
Germ cells are an ideal target for transposons, since these cells pass their genetic material – together with integrated mobile elements – on to all cells of the progeny. But normal body (somatic) cells can also be attacked by transposons. For example, certain viruses carry transposons in their genomes and introduce them into the host cells they infect. Therefore, transposon activity must also be repressed in somatic cells. Recently so-called endo-siRNAs that perform this function were discovered in the fruitfly. A similar class of molecules has been found in mice.
By means of a process called RNA interference, the siRNAs enable the cell to recognize and destroy messenger RNAs derived from transposons. The researchers in Förstemann’s group were able to identify a protein that is essential for the production of endo-siRNAs. It turns out that this is a previously unknown variant of the protein “Loquacious”. In Drosophila, Loquacious can bind to specific RNA molecules that serve as precursors of the endo-siRNAs. Furthermore, the team pinpointed an entirely novel feature of this system: Repression of transposon activity was also detectable when multiple copies of a mobile element were present in the cell but not yet incorporated into the genome.
The phenomenon of RNA interference first came to light only a short time ago, but has already become a well established field of study. Thanks to more recent findings, the known repertoire of small RNAs has grown. As Förstemann stresses, “It is therefore particularly important to discriminate between the various molecular classes in terms of their modes of synthesis and specific functions”. This is no easy task, since all these molecules are similar in size and virtually indistinguishable chemically. “We will now test whether the mechanism we have found in drosophila also exists in mammalian cells. We would also like to know how the mechanism is targeted specifically against sequences present in high copy numbers”.
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Helical Growth of the Arabidopsis Mutant tortifolia2 Does Not Depend on Cell Division Patterns but Involves Handed Twisting of Isolated Cells
28.07.2009
Plant Cell,
2009,
21,
2090-2106, doi:10.1105/tpc.108.061242
published on 28.07.2009
Plant Cell, online article
Plant Cell, online article
Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the {alpha}-tubulin 4 protein. Based on a published high-resolution (2.89 Å) tubulin structure, we predict that Arg-2 of {alpha}-tubulin forms hydrogen bonds with the GTPase domain of {beta}-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.
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mRNA Export - An Integrative Component of Gene Expression
25.07.2009
Nuclear Transport (Ed.: R. Kehlenbach),
2009,
Chapt. 7,
ISBN: 978-1-58706-333-6
published on 25.07.2009
Nuclear Transport, online article
Nuclear Transport, online article
MRNA export is an indispensable step during the complex process of eukaryotic gene expression: the mRNA, created by RNA polymerase II in the nucleus, has to be transported to the cytoplasm, where the ribosomes are responsible for the synthesis of the encoded protein. This transport process is not restricted to the simple passage of the mRNA through the nuclear pore complex, which spans the nuclear envelope, but is embedded into the gene expression pathway. During transcription, the message is capped, spliced and polyadenylated and mRNA export factors are loaded onto the nascent transcript. This maturation and assembly of the mRNA into a mature messenger ribonucleoprotein particle is controlled by nuclear surveillance systems: the nuclear exosome and the Mlp1‑2 system prevent the escape of aberrant transcripts to the cytoplasm. Only correctly assembled mRNPs are transported through the nuclear pore to the cytoplasm by the mRNA export receptor Mex67‑Mtr2/Tap‑p15, which is recruited to the mRNA by interaction with the mRNP‑bound TREX complex and SR proteins. This tight coupling between the single steps of the nuclear gene expression guarantees an efficient and accurate transfer of the genetic information to the cytoplasm.
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Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex
20.07.2009
Nucl. Acids Res.,
2009,
1-7, doi:10.1093/nar/gkp601
published on 20.07.2009
Nucl. Acids Res., online article
Nucl. Acids Res., online article
Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop β10-β11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation–elongation transition.
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Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II
28.06.2009
Nature Genetics,
2009,
41,
936-940, doi:10.1038/ng.405
published on 28.06.2009
Nature Genetics, online article
Nature Genetics, online article
Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance5. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants4, 6. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.
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Structural Basis of Transcription: Mismatch-Specific Fidelity Mechanisms and Paused RNA Polymerase II with Frayed RNA
26.06.2009
Mol. Cell,
2009,
34(6),
710-721, doi:10.1016/j.molcel.2009.06.002
published on 26.06.2009
Mol. Cell, online article
Mol. Cell, online article
We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNA⋅RNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a T⋅U mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3′ end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3′ nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.
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Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2.
25.06.2009
EMBO J.,
2009,
28(15),
2293-2306, doi:10.1038/emboj.2009.175
published on 25.06.2009
EMBO J., online article
EMBO J., online article
Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated -helical and -hairpin elements to the UPF1 CH-domain. The -helical region binds sixfold more weakly than the -hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.
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Fast and accurate automatic structure prediction with HHpred
13.06.2009
Proteins,
2009,
77(S9),
128-132, doi:10.1002/prot.22499
published on 13.06.2009
Proteins, online article
Proteins, online article
Automated protein structure prediction is becoming a mainstream tool for biological research. This has been fueled by steady improvements of publicly available automated servers over the last decade, in particular their ability to build good homology models for an increasing number of targets by reliably detecting and aligning more and more remotely homologous templates. Here, we describe the three fully automated versions of the HHpred server that participated in the community-wide blind protein structure prediction competition CASP8. What makes HHpred unique is the combination of usability, short response times (typically under 15 min) and a model accuracy that is competitive with those of the best servers in CASP8.
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An Unfolded CH1 Domain Controls the Assembly and Secretion of IgG Antibodies
12.06.2009
A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the CH1 domain of the heavy chain is intrinsically disordered invitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain CL domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the CH1 domain and competes for binding to the CL domain. In vivo experiments demonstrate that requirements identified for folding the CH1 domain invitro, including association with a folded CL domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.
Adhesion Dance with Raver
10.06.2009
Structure,
2009,
17(6),
781-783, doi:10.1016/j.str.2009.05.004
published on 10.06.2009
Structure, online article
Structure, online article
The localization of mRNAs in subcellular compartments is an efficient way to spatially restrict gene expression. Crystal structures of raver1-vinculin reported by Izard and coworkers now suggest a possible mechanism for mRNA localization during the assembly of focal adhesions.
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Breaking the Dogma of the Metal-Coordinating Carboxylate Group in Integrin Ligands: Introducing Hydroxamic Acids to the MIDAS To Tune Potency and Selectivity
02.06.2009
Angewandte Chemie,
2009,
48, 24,
4436 - 40
published on 02.06.2009
Angewandte Chemie, online article
Angewandte Chemie, online article
A suitable substitute: All integrin receptors bind their ligands, which contain an aspartate residue, in the metal-ion- dependent adhesion site (MIDAS). So far all attempts to replace the carboxyl group of aspartate with other, pharmacologically favorable isosteric groups have failed. Now it has been shown that a hydroxamic acid group can replace the carboxyl group; the resulting ligand retains its high binding activity. The picture shows one such ligand in the binding site of v3.
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Ligands for Mapping αvβ3-Integrin Expression in Vivo
02.06.2009
Accounts of Chemical Research,
2009,
42, 7,
969 - 80
published on 02.06.2009
Accounts of Chemical Research online article
Accounts of Chemical Research online article
α5β1-integrins play a key role in angiogenesis, the formation of new vessels in tissues that lack them. By serving as receptors for a variety of extracellular matrix proteins containing an arginine-glycine-aspartic acid (RGD) sequence, these integrins mediate migration of endothelial cells into the basement membrane and regulate their growth, survival, and differentiation. Besides being involved in angiogenesis, the αvβ3-integrin is also presented on tumor cells of various origin, where it is involved in the processes that govern metastasis.
Because the αvβ3-integrin is an attractive target for cancer treatment, high-affinity ligands containing the RGD sequence, for example, cyclic pentapeptides, have been developed. They inhibit angiogenesis, induce endothelial apoptosis, decrease tumor growth, and reduce invasiveness and spread of metastasis. This development finally resulted in cyclo(RGDf(NMe)V) (cilengitide), which is a drug for the treatment of glioblastoma (currently in phase III clinical trials).
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A movie of the RNA polymerase nucleotide addition cycle
27.05.2009
Curr. Opin. Struct. Biol.,
2009,
19(3),
294-299, doi:10.1016/j.sbi.2009.04.005
published on 27.05.2009
Curr. Opin. Struct. Biol., online article
Curr. Opin. Struct. Biol., online article
During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop. The movie facilitates teaching and a mechanistic analysis of transcription and can be downloaded from http://www.lmb.uni-muenchen.de/cramer/pr-materials.
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Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain
26.05.2009
J. Mol. Biol.,
2009,
389(1),
211-225, doi:10.1016/j.jmb.2009.04.016
published on 26.05.2009
J. Mol. Biol., online article
J. Mol. Biol., online article
During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.
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HHomp—prediction and classification of outer membrane proteins
08.05.2009
Nucleic Acids Research,
2009,
37,
W446-W451, doi:10.1093/nar/gkp325
published on 08.05.2009
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Outer membrane proteins (OMPs) are the transmembrane proteins found in the outer membranes of Gram-negative bacteria, mitochondria and plastids. Most prediction methods have focused on analogous features, such as alternating hydrophobicity patterns. Here, we start from the observation that almost all β-barrel OMPs are related by common ancestry. We identify proteins as OMPs by detecting their homologous relationships to known OMPs using sequence similarity. Given an input sequence, HHomp builds a profile hidden Markov model (HMM) and compares it with an OMP database by pairwise HMM comparison, integrating OMP predictions by PROFtmb. A crucial ingredient is the OMP database, which contains profile HMMs for over 20 000 putative OMP sequences. These were collected with the exhaustive, transitive homology detection method HHsenser, starting from 23 representative OMPs in the PDB database. In a benchmark on TransportDB, HHomp detects 63.5% of the true positives before including the first false positive. This is 70% more than PROFtmb, four times more than BOMP and 10 times more than TMB-Hunt. In Escherichia coli, HHomp identifies 57 out of 59 known OMPs and correctly assigns them to their functional subgroups.
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Non-Hydrolyzable RNA-Peptide Conjugates: A Powerful Advance in the Synthesis of Mimics for 3-Peptidyl tRNA Termini
24.04.2009
Angew. Chem. Int. Ed.,
2009,
48(42),
4056-4060, doi:10.1002/anie.200900939
published on 24.04.2009
Angew. Chem. Int. Ed., online article
Angew. Chem. Int. Ed., online article
Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA-peptide conjugates that mimic peptidyl-tRNA would be desirable, especially for ribosome structural biology. A flexible solid-phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.
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The HSP90 binding mode of a radicicol-like E-oxime determined by docking, binding free energy estimations, and NMR 15N chemical shifts
15.04.2009
Biophysical Chemistry,
2009,
143 Issue 3,
111-23
published on 15.04.2009
Biophysical Chemistry, online article
Biophysical Chemistry, online article
We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses
are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone 15N provides an additional evaluation criteria. As a final test we check the binding modes against
available structure–activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol–HSP90 complex.
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Interaction of human heat shock protein 70 with tumor-associated peptides
01.04.2009
Biological Chemistry,
2009,
390 , 4,
305- 12
published on 01.04.2009
Biological Chemistry, online article
Biological Chemistry, online article
Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immuno-logical approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (Kd=0.58 [mu]M at 25[degrees]C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (Kd=32 [mu]M) and melanoma antigen recognized by T cells (MART-1) (Kd=2.4 mM). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (Kd=0.04 mM) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.
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Sequence context-specific profiles for homology searching
10.03.2009
Sequence alignment and database searching are essential tools in
biology because a protein’s function can often be inferred from
homologous proteins. Standard sequence comparison methods
use substitution matrices to find the alignment with the best sum
of similarity scores between aligned residues. These similarity
scores do not take the local sequence context into account. Here,
we present an approach that derives context-specific amino acid
similarities from short windows centered on each query sequence
residue. Our results demonstrate that the sequence context contains
much more information about the expected mutations than
just the residue itself. By employing our context-specific similarities
(CS-BLAST) in combination with NCBI BLAST, we increase the
sensitivity more than 2-fold on a difficult benchmark set, without
loss of speed. Alignment quality is likewise improved significantly.
Furthermore, we demonstrate considerable improvements when
applying this paradigm to sequence profiles: Two iterations of
CSI-BLAST, our context-specific version of PSI-BLAST, are more
sensitive than 5 iterations of PSI-BLAST. The paradigm for biological
sequence comparison presented here is very general. It can replace
substitution matrices in sequence- and profile-based alignment and
search methods for both protein and nucleotide sequences.
Structure and Ligand Binding of the Extended Tudor Domain of D. melanogaster Tudor-SN
14.02.2009
Journal of Molecular Biology,
2009,
387(4),
921-934, doi:10.1016/j.jmb.2009.02.018
published on 14.02.2009
Journal of Molecular Biology, online article
Journal of Molecular Biology, online article
The Tudor-SN protein (p100, SND1) has been implicated in a variety of cellular processes, such as transcription, processing of edited double-stranded RNA, and splicing regulation. Molecular details of these functions are not yet understood. Tudor domains have previously been shown to bind methylated ligands, such as methylated lysines and arginines. It has been suggested that the role of Tudor-SN in splicing may involve binding to such methylated ligands or to the methylated 5′ cap of spliceosomal snRNAs.
Here, we report the crystal structure of the extended Tudor domain of Tudor-SN from Drosophila melanogaster to a resolution of 2.1 Å. NMR secondary chemical shifts, relaxation data, and residual dipolar couplings indicate that the solution and crystal structures are similar. Binding of various ligands was investigated by NMR. Binding sites and affinities were characterized by chemical shift perturbations. We show that the aromatic cage of the Tudor domain specifically binds a peptide containing symmetrically dimethylated arginines (sDMA) with micromolar affinity, while the same peptide comprising nonmethylated arginines does not show significant chemical shift perturbations. Tudor-SN preferentially recognizes sDMA over asymmetrically dimethylated arginine (aDMA). In contrast, two 5′ cap analogues with different methylation patterns, as well as mono-, di-, and trimethyllysines, show no binding.
Our data demonstrate that the Tudor domain of Tudor-SN specifically recognizes sDMA-containing ligands. The aromatic cage of Tudor-SN is very similar to the one in the Tudor domain of the survival of motor neuron protein, which also recognizes sDMA peptides, indicating a conserved binding motif for this methylation mark. Recognition of sDMA in the C-terminal tails of spliceosomal Sm proteins suggests how Tudor-SN may interact with small nuclear ribonucleoprotein particles during the regulation of splicing.
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The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA
10.02.2009
Nucleic Acids Res,
2009,
doi:10.1093/nar/gkp059,
1-12
published on 10.02.2009
Nucleic Acids Research, online article
Nucleic Acids Research, online article
RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-Ilike receptor, LGP2, differentially regulates RIG-Iand MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal
regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5’-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6A ° crystal structure of LGP2 RD along with in vitro and in vivo functional analyses
and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5’-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for
dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.
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Impact of Order and Disorder in RGD Nanopatterns on Cell Adhesion
10.02.2009
We herein present a novel platform of well-controlled ordered and disordered nanopatterns positioned with a cyclic peptide of arginine-glycine-aspartic acid (RGD) on a bioinert poly(ethylene glycol) background, to study whether the nanoscopic order of spatial patterning of the integrin-specific ligands influences osteoblast adhesion. This is the first time that the nanoscale order of RGD ligand patterns was varied quantitatively, and tested for its impact on the adhesion of tissue cells. Our findings reveal that integrin clustering and such adhesion induced by RGD ligands is dependent on the local order of ligand arrangement on a substrate when the global average ligand spacing is larger than 70 nm; i.e., cell adhesion is “turned off” by RGD nanopattern order and “turned on” by the RGD nanopattern disorder if operating at this range of interligand spacing.
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Structural basis for competitive interactions of Pex14 with the import receptors Pex5 and Pex19
05.02.2009
Protein import into peroxisomes depends on a complex and dynamic network of protein–protein interactions. Pex14 is a central component of the peroxisomal import machinery and binds the soluble receptors Pex5 and Pex19, which have important function in the assembly of peroxisome matrix and membrane, respectively. We show that the N-terminal domain of Pex14, Pex14(N), adopts a three-helical fold. Pex5 and Pex19 ligand helices bind competitively to the same surface in Pex14(N) albeit with opposite directionality. The molecular recognition involves conserved aromatic side chains in the Pex5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19. The Pex14–Pex5 complex structure reveals molecular details for a critical interaction in docking Pex5 to the peroxisomal membrane. We show that mutations of Pex14 residues located in the Pex5/Pex19 binding region disrupt Pex5 and/or Pex19 binding in vitro. The corresponding full-length Pex14 variants are impaired in peroxisomal membrane localisation in vivo, showing that the molecular interactions mediated by the N-terminal domain modulate peroxisomal targeting of Pex14.
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Structure–function studies of the RNA polymerase II elongation complex
01.02.2009
Acta Chrystallogr. D,
2009,
65(2),
112-120, doi:10.1107/S0907444908039875
published on 01.02.2009
Acta Chrystallogr. D, online article
Acta Chrystallogr. D, online article
RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.
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Time-Resolved Binding of Azithromycin to Escherichia coli Ribosomes
30.01.2009
J. Mol. Biol.,
2009,
385(4),
1179-1192, doi:10.1016/j.jmb.2008.11.042
published on 30.01.2009
J. Mol. Biol., online article
J. Mol. Biol., online article
Azithromycin is a semisynthetic derivative of erythromycin that inhibits bacterial protein synthesis by binding within the peptide exit tunnel of the 50S ribosomal subunit. Nevertheless, there is still debate over what localization is primarily responsible for azithromycin binding and as to how many molecules of the drug actually bind per ribosome. In the present study, kinetic methods and footprinting analysis are coupled together to provide time-resolved details of the azithromycin binding process. It is shown that azithromycin binds to Escherichia coli ribosomes in a two-step process: The first-step involves recognition of azithromycin by the ribosomal machinery and places the drug in a low-affinity site located in the upper part of the exit tunnel. The second step corresponds to the slow formation of a final complex that is both much tighter and more potent in hindering the progression of the nascent peptide through the exit tunnel. Substitution of uracil by cytosine at nucleoside 2609 of 23S rRNA, a base implicated in the high-affinity site, facilitates the shift of azithromycin to this site. In contrast, mutation U754A hardly affects the binding process. Binding of azithromycin to both sites is hindered by high concentrations of Mg2+ ions. Unlike Mg2+ ions, polyamines do not significantly affect drug binding to the low-affinity site but attenuate the formation of the final complex. The low- and high-affinity sites of azithromycin binding are mutually exclusive, which means that one molecule of the drug binds per E. coli ribosome at a time. In contrast, kinetic and binding data indicate that in Deinococcus radiodurans, two molecules of azithromycin bind cooperatively to the ribosome. This finding confirms previous crystallographic results and supports the notion that species-specific structural differences may primarily account for the apparent discrepancies between the antibiotic binding modes obtained for different organisms.
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Less Is More for Leaderless mRNA Translation
30.01.2009
Mol. Cell,
2009,
33(2),
141-142, doi:10.1016/j.molcel.2009.01.006
published on 30.01.2009
Mol. Cell, online article
Mol. Cell, online article
In this issue of Molecular Cell, Kaberdina et al. (2009) show that prolonged exposure of ribosomes to the antibiotic kasugamycin triggers the spontaneous loss of small subunit proteins and produces a reduced ribosomal particle that exclusively translates leaderless mRNAs.
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The Native 3D Organization of Bacterial Polysomes
23.01.2009
Recent advances have led to insights into the structure of the bacterial ribosome, but little is knownabout the 3D organization of ribosomes in the context of translating polysomes. We employed cryoelectron tomography and a template-matching approach to
map 70S ribosomes in vitrified bacterial translation extracts and in lysates of active E. coli spheroplasts. In these preparations, polysomal arrangements were observed inwhich neighboring ribosomesare densely packed and exhibit preferred orientations. Analysis of characteristic examples of polysomes reveals a staggered or pseudohelical organization of ribosomes
along the mRNA trace, with the transcript being sequestered on the inside, the tRNA entrance sites being accessible, and the polypeptide exit sites facing the cytosol. Modeling of elongating nascent polypeptide chains suggests that this arrangement maximizes the distance between nascent chains on adjacent
ribosomes, thereby reducing the probability of intermolecular
interactions thatwould give rise to aggregation and limit productive folding.
A Distinct Mode of Interaction of a Novel Ketolide Antibiotic That Displays Enhanced Antimicrobial Activity
21.01.2009
Antimicrob. Agents Ch.,
2009,
53(4),
1411-1419, doi:10.1128/AAC.01425-08
published on 21.01.2009
Antimicrob. Agents Ch., online article
Antimicrob. Agents Ch., online article
Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin resistant strains, while maintaining their activity against erythromycin susceptible ones. In this study we present a new ketolide K-1325 that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild type E.coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the aryl-alkyl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increase affinity of U2609C mutant ribosomes for the drug, and rationalizing the enhanced effectiveness of this new ketolide.
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A Boost for Sequence Searching
01.10.2009
GIT Laboratory Journal,
2009,
9-10,
22-23
published on 10.01.2009
GIT Laboratory Journal, online article
GIT Laboratory Journal, online article
With over 47,000 citations, the sequence search method BLAST has been an essential tool in biological research since its development in 1990. By accounting for the influence of sequence context on the mutation probabilities of amino acids, context-specific BLAST (CS-BLAST) achieves two-fold higher sensitivity for distantly related protein sequences at the same speed and error rate.
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Thioredoxin as a fusion tag for carrier-driven crystallization
02.01.2009
Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion
proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a fourresidue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that
can aid crystallization of difficult targets.
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Cytosolic Viral Sensor RIG-I Is a 5′-Triphosphate–Dependent Translocase on Double-Stranded RNA
20.02.2009
RIG-I is a cytosolic multi-domain protein that detects viral RNA and elicits an antiviral immune response. Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal and the regulatory domain prevents signaling in the absence of viral RNA. 5’-triphosphate and double stranded (ds) RNA are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA. However, the function of the ATPase domain that is also required for activity is less clear. Using single-molecule fluorescence assays we
discovered a robust, ATP-powered dsRNA translocation activity of RIG-I. The CARDs dramatically suppress translocation in the absence of 5’-triphosphate and the activation by 5’-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis. This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses.
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