Content
2007
Functional Architecture of RNA Polymerase I
27.12.2007
Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A° cryoelectron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 30-RNA cleavage activity, which requires the C-terminal
domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 30-end
trimming.
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The membrane-integrated transcriptional activator CadC of Escherichia coli senses lysine indirectly via the interaction with the lysine permease LysP
16.12.2007
Molecular Microbiology,
2007,
67 (3),
570-83
published on 16.12.2007
Molecular Microbiology, online article
Molecular Microbiology, online article
In an acidic (pH 5.8) and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, catalysing the decarboxylation of lysine to cadaverine, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family and directly binds to the DNA in the cadBA promoter region. Here we describe that CadC
senses the extracellular lysine not directly but indirectly requiring the interplay with the lysine permease LysP. Biochemical analyses of isolated CadC or the periplasmic domain of CadC (CadC188–512) revealed an unexpectedly low affinity for lysine, making it unlikely that CadC is a direct sensor for this substrate. Moreover, CadC hybrid proteins, in which the transmembrane domain or single amino acids were replaced, supported lysine-independent cadBA expression but retained a pH-dependent regulation. These CadC mutants were resistant to the effect of an overproduction of LysP, which represses cadBA
expression in wild-type cells. Our results suggest a model according to which CadC is inactivated by an interaction with LysP at a low external lysine concentration. When lysine is abundantly available, the interaction between LysP and CadC is released,
and CadC becomes susceptible to activation by low pH.
Site specific phosphorylation of yeast RNA polymerase I
15.12.2007
Nucleic Acids Research,
2007,
doi: 10.1093/nar/gkm1093,
793-802
published on 15.12.2007
Nucleic Acids Research, online article
Nucleic Acids Research, online article
All nuclear RNA polymerases are phosphoprotein complexes. Yeast RNA polymerase I (Pol I) contains approximately 15 phosphate groups, distributed to 5 of the 14 subunits. Information about the function of the single phosphosites and their position in the primary, secondary and tertiary structure is lacking. We used a rapid and efficient way to purify yeast RNA Pol I to determine 13 phosphoserines and –threonines. Seven of these phosphoresidues could be located in the 3D-homology model for Pol I, five of them are more at the surface. The single phosphorylated residues were systematically mutated and the resulting strains and Pol I preparations were analyzed in cellular growth, Pol I composition, stability and genetic interaction with non- essential components of the transcription machinery. Surprisingly, all Pol I phosphorylations analyzed were found to be non-essential post-translational modifications. However, one mutation (subunit A190 S685D) led to higher growth rates in the presence of 6AU or under environmental stress
conditions, and was synthetically lethal with a deletion of the Pol I subunit A12.2, suggesting a role in RNA cleavage/elongation or termination. Our results suggest that individual major or constitutively phosphorylated residues contribute to non-essential Pol I-functions.
2008 Gottfried Wilhelm Leibniz Prize for CIPSM researcher Elena Conti
07.12.2007
published on 07.12.2007
www.dfg.de
www.dfg.de
The Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) announced yesterday the winners of its 2008 Gottfried Wilhelm Leibniz Prize. At its meeting on December 6th 2007, the DFG Joint Committee named CIPSM researcher Dr. Elena Conti as recipient of Germany's most highly endowed research award. Elena will receive 1.25 million euros flexibly over a period of seven years to finance her research.
Dr. Elena Conti (40) works in research area C, Protein Structure and Networks in the field of biochemistry at CIPSM.
Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins
04.12.2007
Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a threedimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chlororibosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.
Molecular basis of RNA-dependent RNA polymerase II activity
15.11.2007
RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene tran- scription. There is, however, evidence that Pol II also possesses RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA template, can extend RNA by several nucleotides in the absence of DNA, and has been implicated in the replication of the RNA genomes of hepatitis delta virus (HDV) and plant viroids. Here we show the intrinsic RdRP activity of Pol II with only pure polymerase, an RNA template- product scaffold and nucleoside triphosphates (NTPs). Crystallography reveals the template-product duplex in the site occupied by the DNA-RNA hybrid during transcription. RdRP activity resides at the active site used during transcription, but it is slower and less processive than DNA-dependent activity. RdRP activity is also obtained with part of the HDV antigenome. The complex of transcription factor IIS (TFIIS) with Pol II can cleave one HDV strand, create a reactive stem-loop in the hybrid site, and extend the new RNA 3' end. Short RNA stem-loops with a 5' extension suffice for activity, but their growth to a critical length apparently impairs processivity. The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes.
Allowing an elite
15.11.2007
"There's great excitement here"
“If the show is over in five years, then it was but a flash in the pan”
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Seeing Cisplatin - Discerning how an anticancer drug meddles with replication, transcription
12.11.2007
Chemical & Engineering News,
2007,
Vol. 85, Nr. 46,
13
published on 12.11.2007
Chemical & Engineering News, online article
Chemical & Engineering News, online article
CISPLATIN HAS BEEN USED to treat cancer for decades. But the drug's detailed molecular interactions with the enzymes it aims to interrupts have remained obscure. In two separate papers, researchers are now reporting the first crystal structures of cisplatin complexed with two key enzymes.
"For the first time, we are getting deep insight on what goes on at the atomic level with cisplatin and cellular machinery," says Thomas Carell, a protein chemist at the University of Munich.
Cisplatin creates bulky platinum adducts with pairs of guanidine bases that are side-by-side or one base apart on genomic DNA. The extra bulk blocks most DNA polymerases that slide along DNA when they copy the genetic code during cell division. The adducts also obstruct RNA polymerases, which transcribe DNA to make proteins. But no structures of cisplatin in complex with either DNA or RNA polymerases have come to light until now.
In one report, Carell and Karl-Peter Hopfner announce the structure of a renegade DNA polymerase called Pol η. Normal DNA polymerases get stalled by cisplatin because their grip on DNA is so tight that there's no room for adduct riffraff. But Pol η manages to replicate over unwelcome DNA adducts.
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Original Science article, Science 2007, 318, 967
See also under www.cipsm.de
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Mechanism of transcriptional stalling at cisplatindamaged DNA
11.11.2007
Nature Structural & Molecular Biology,
2007,
doi:10.1038/nsmb1314,
1-7
published on 11.11.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II)
and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion
in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP
misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according
to an ‘A-rule’ known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation
barrier, even in the presence of a G.A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism
differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated
misincorporation that blocks transcription.
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Structural Aspects of RbfA Action during Small Ribosomal Subunit Assembly
09.11.2007
Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S.RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA’s functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved inmRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA duringmaturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.
Bypass of DNA Lesions Generated During Anticancer Treatment with Cisplatin by DNA Polymerase Eta
09.11.2007
DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UVinduced cis–syn thymine dimers. We present structural and biochemical analysis of how Pol h copies Pt-GG–containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.
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Press commentaries:
Lübecker Nachrichten www.LN-online.de
Other articles can be found under www.handelsbaltt.de, www.Pharmazeutische-Zeitung.de and Frankfurter Neue Presse by searching for Carell or Hopfner
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Multiple N-Methylation by a Designed Approach Enhances Receptor Selectivity
01.11.2007
Journal of Medicinal Chemistry,
2007,
50 (24),
5878-81
published on 01.11.2007
Journal of Medicinal Chemistry, online article
Journal of Medicinal Chemistry, online article
An unselective cyclic peptide integrin ligand was sequentially
N-methylated by a designed approach, where only the externally
oriented (solvent exposed) amide bonds were N-methylated. The
N-methylation resulted in tremendous enhancement in selectivity among the different integrin receptor subtypes (alpha5beta1, alphavbeta3, and alphaIIbbeta3). Conformational and docking studies were performed, which suggested that the receptor selectivity is principally caused by reduced backbone flexibility due to N-methylation.
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Archaeal transcription: function of an alternative transcription factor B from Pyrococcus furiosus
26.10.2007
J. Bacteriol.,
2007,
doi:10.1128/JB.01498-07,
published on 26.10.2007
The Journal of Bacteriology, online article
The Journal of Bacteriology, online article
The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) has been characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter dependent transcription initiation, but photochemical cross-linking experiments indicate that transcription complexes formed with TFB2 at the strong gdh promoter are similar in orientation and occupancy to those formed by TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a pre-formed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 show that the low activity of TFB2 is determined mainly by its N-terminus. The low activity of TFB2 in promoter opening and transcription can be partly relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn-ribbon and B-finger motifs, is important in promoter opening, and that TFE can compensate for defects in the N-terminus through enhancement of promoter opening.
Structural Biology of RNA Polymerase III: Mass Spectrometry Elucidates Subcomplex Architecture
16.10.2007
RNA polymerases (Pol) II and III synthesize eukaryotic mRNAs and tRNAs, respectively. The crystal structure of the 12 subunit Pol II is known, but only limited structural information is available for the 17 subunit Pol III. Using mass spectrometry (MS), we correlated masses of Pol II complexes with the Pol II structure. Analysis of Pol III showed that the complete enzyme contains a single copy of each subunit and revealed a 15 subunit form lacking the Pol III-specific subcomplex C53/37. DMSO treatment dissociated the C17/25 heterodimer of Pol III, confirming a peripheral location as its counterpart in Pol II. Tandem MS revealed the Pol III-specific subunits C82 and C34 dissociating as a heterodimer. C11 was retained, arguing against a stable trimeric subcomplex, C53/37/11. These data suggest that Pol III consists of a 10 subunit Pol II-like core; the peripheral heterodimers C17/25, C53/37, and C82/34; and subunit C31, which bridges between C82/34, C17/25, and the core.
J-ONLY-TOCSY: Efficient suppression of RDC-induced transfer in homonuclear TOCSY experiments using JESTER-1-derived multiple pulse sequences
20.09.2007
Journal of Magnetic Resonance,
2007,
189,
217-27
published on 20.09.2007
Journal of Magnetic Resonance, online article
Journal of Magnetic Resonance, online article
The main purpose of homonuclear Hartmann–Hahn or TOCSY experiments is the assignment of spin systems based on efficient
coherence transfer via scalar couplings. In partially aligned samples, however, magnetization is also transferred via residual dipolar
couplings (RDCs) and therefore through space correlations can be observed in COSY and TOCSY experiments that make the
unambiguous assignment of covalently bound spins impossible. In this article, we show that the JESTER-1 multiple pulse sequence, originally designed for broadband heteronuclear isotropic Hartmann–Hahn transfer, efficiently suppresses the homonuclear dipolar coupling Hamiltonian. This suppression can be enhanced even further by variation of the supercycling scheme. The application of the resulting element in homonuclear TOCSY periods results in coherence transfer via J-couplings only. As a consequence, the assignment of scalar coupled spin systems is also possible in partially aligned samples. The bandwidth of coherence transfer for the JESTER-1-derived sequences is comparable to existing TOCSY multiple pulse sequences. Results are demonstrated in theory and experiment.
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Specific Integrin Labeling in Living Cells Using Functionalized Nanocrystals
20.08.2007
Quantum dot (QD) bioimaging has recently been described
as the most exciting new technique to emerge from the collaboration of physicists and biologists. This new molecular
imaging technology is based on the selective fluorescent labeling of biological molecules. QDs are fluorescent nanocrystals showing a wide-range absorption spectrum (400–650 nm) and a narrow, symmetric emission spectrum. By choosing the appropriate QD size, the emission wavelength can be tuned even to the near infrared. This allows the QD signal to be clearly distinguished from the cell autofluorescence background. Finally, the stability of QDs against oxidation and photobleaching makes them highly suitable for single-molecule labeling compared to conventional dyes. Based on these unique spectral, physical, and chemical properties, QDs are extremely useful as a technique to “light up” biological events. However, besides all their advantages, QDs do not offer a continuous fluorescence signal as they exhibit a statistical blinking behavior.
This feature hampers automated image processing and might adulterate the quantitative parameters that are extracted from the trajectories of such blinking particles.
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High-Resolution Structure of a Na+/H+ Antiporter Dimer Obtained by Pulsed Electron Paramagnetic Resonance Distance Measurements
17.08.2007
Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na1/H1 antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.
Development of a Peptidomimetic Ligand for Efficient Isolation and Purification of Factor VIII via Affinity Chromatography
11.08.2007
The Journal of Cell Biology,
2007,
50,
4329-39
published on 11.08.2007
Journal of Medicinal Chemistry, online article
Journal of Medicinal Chemistry, online article
Hemophilia A, one of the most severe bleeding disorders, results from an inherited deficiency of factor VIII (FVIII) function. Treatment by injection of FVIII has been a common procedure for decades. Nevertheless, the production and purification of FVIII remains a challenging task. Current procedures using immunoaffinity
chromatography are expensive and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified by use of octapeptide ligands, but their low protease-resistance limits their application. We here report the systematic rational and combinatorial optimization procedure that allowed us to transfer the octapeptide ligands into a small peptidomimetic. This compound is the smallest ligand known for separation of such a large protein (330 kDa). It not only binds and purifies FVIII with high efficiency but also is stable, protease-resistant, and cheap to produce in preparative scale. Hence it offers a valuable alternative to antibody-based purification procedures.
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Finding the right spot to start transcription
01.08.2007
Nature Structural & Molecular Biology,
2007,
14 (8),
686-7
published on 01.08.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
How does RNA polymerase II cooperate with initiation factors to locate transcription start sites throughout the genome? A new cross-linking approach reveals previously unknown initiation factor–binding sites on the polymerase surface. The resulting model of the transcription initiation complex suggests that initiation factors cooperate above and inside the polymerase active center cleft to open DNA and find the transcription start site. In all living cells, the expression of genetic programs is governed by the dynamic transcriptome, which is established mainly by regulated transcription initiation at gene promoters. Thus, if we want to understand gene regulation, we must know the mechanism of transcription initiation. Initiation by RNA polymerase II (Pol II), which catalyzes eukaryotic messenger RNA transcription, requires the general transcription factors TFIIB, TFIID (containing the TATA box–binding protein, TBP), TFIIE, TFIIF and TFIIH, which assemble with the polymerase into a preinitiation complex (PIC) at the promoter1–4. The structure of the large and complex PIC remains under debate even at a topological level5. On page 696 of this
issue, new work from Chen et al. of the Hahn laboratory6 provides insights into the PIC architecture and demonstrates an approach for elucidating the topology of a transient multicomponent complex even when only partial structural information is available.
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Drosophilia microRNAs Are Sorted into Functionally Distinct Argonaute Complexes after Production by Dicer-1
27.07.2007
Cell,
2007,
130,
287-97
published on 27.07.2007
online article
online article
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RICSs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophilia, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Here, we show that despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNAs-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2.
Multisubunit RNA Polymerases Melt Only a Single DNA Base Pair Downstream of the Active Site
27.07.2007
J. Biol. Chem.,
2007,
282,
21578-82
published on 27.07.2007
Journal of Biological Chemistry, online article
Journal of Biological Chemistry, online article
During gene transcription, RNA polymerase (Pol) II
encounters obstacles, including lesions in the DNA template.
Here, we review a recent structure–function analysis of Pol II
transcribing DNA with a bulky photo-lesion in the template
strand. The study provided the molecular basis for recognition
of a damaged DNA by Pol II, which is the first step in transcription-
coupled DNA repair (TCR). The results have general implications
for damage recognition and the TCR mechanism.
piRNAs-the ancient hunters of genome invaders
16.07.2007
Genes & Development,
2007,
21,
1707-13
published on 16.07.2007
online article
online article
In addition to miRNAs and siRNAs, a third small RNA silencing system has been uncovered that prevents the spreading of selfish genetic elements. Production of the Piwi-associated RNAs (piRNAs), which mediate the silencing activity in this pathway, is initiated at a few master control regions within the genome. The nature of the primary piRNA-generating transcript is still unknown, but RNA interference (RNAi)-like cleavage events are likely defining the 5'-ends of mature piRNAs. We summarize the recent literature on piRNA biogenesis and function with an emphasis on work in Drosophila, where genetics and biochemistry have met very successfully.
The RGD motif in fi bronectin is essential for development but dispensable for fibril assembly
02.07.2007
The Journal of Cell Biology,
2007,
178 (1),
167-78
published on 02.07.2007
Journal of Cell Biology, online article
Journal of Cell Biology, online article
Fibronectin (FN) is secreted as a disulfi de-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fi brils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–defi cient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise
assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FNRGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our fi ndings demonstrate that FN contains a novel motif for integrin binding and fi bril formation whose activity is controlled by amino acid modifi cation.
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U2AF-homology motif interactions are required for alternative splicing regulation by SPF45
02.07.2007
Nature Structural & Molecular Biology,
2007,
14 (7),
620-29
published on 02.07.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3' splice site– recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-Armström crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.
Structural basis for DNA duplex sepration by a superfamily-2 helicase
01.07.2007
Nature Structural & Molecular Biology,
2007,
14,
647-52
published on 01.07.2007
online article
online article
To reveal the mechanism of processive strand separation by superfamily-2 (SF2) 3'-5' helicases, we determined apo and DNA-bound crystal structures of archaeal Hel308, a helicase that unwinds lagging strands and is related to human DNA polymerase h.
Our structure captures the duplex-unwinding reaction, shows that initial strand separation does not require ATP and identifies a
prominent beta-hairpin loop as the unwinding element. Similar loops in hepatitis C virus NS3 helicase and RNA-decay factors
support the idea that this duplex-unwinding mechanism is applicable to a broad subset of SF2 helicases. Comparison with ATP-bound
SF2 enzymes suggests that ATP promotes processive unwinding of 1 base pair by ratchet-like transport of the 3' product
strand. Our results provide a first structural framework for strand separation by processive SF2 3'-5' helicases and reveal
important mechanistic differences from SF1 helicases.
DNA photodamage recognition by RNA polymerase II
19.06.2007
During gene transcription, RNA polymerase (Pol) II
encounters obstacles, including lesions in the DNA template.
Here, we review a recent structure–function analysis of Pol II
transcribing DNA with a bulky photo-lesion in the template
strand. The study provided the molecular basis for recognition
of a damaged DNA by Pol II, which is the first step in transcription-
coupled DNA repair (TCR). The results have general implications
for damage recognition and the TCR mechanism.
CPD Damage Recognition by Transcribing RNA Polymerase II
09.02.2007
Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of RNA polymerase (Pol) II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine misincorporation into mRNA, which blocks translocation. Artificial replacement of the uridine by adenosine enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II.
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