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1.
While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family. 相似文献
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Nozomi Asano Koji Kato Akiyoshi Nakamura Keisuke Komoda Isao Tanaka Min Yao 《Nucleic acids research》2015,43(9):4746-4757
Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The long α-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor. 相似文献
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Yang W Chen WY Wang H Ho JW Huang JD Woo PC Lau SK Yuen KY Zhang Q Zhou W Bartlam M Watt RM Rao Z 《Nucleic acids research》2011,39(22):9803-9819
Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg2+ or Mn2+ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg2+ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases. 相似文献
4.
Boehringer D O'Farrell HC Rife JP Ban N 《The Journal of biological chemistry》2012,287(13):10453-10459
The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. 相似文献
5.
Despite decades of its use in diabetes research, the mechanism of cytotoxicity of streptozotocin (STZ) toward pancreatic β-islet cells has remained a topic of discussion. Although STZ toxicity is likely a function of its capacity to promote DNA alkylation, it has been proposed that STZ induces pancreatic β-cell death through O-GlcNAcase inhibition. In this report, we explore the binding mode of STZ to a close homolog of human O-GlcNAcase, BtGH84 from Bacteroides thetaiotaomicron. Our results show that STZ binds in the enzyme active site in its intact form, without the formation of a covalent adduct, consistent with solution studies on BtGH84 and human O-GlcNAcase, as well as with structural work on a homolog from Clostridium perfringens. The active site of the BtGH84 is considerably deformed upon STZ binding and as a result the catalytic machinery is expelled from the binding cavity. 相似文献
6.
Avinash S. Punekar Josefine Liljeruhm Tyson R. Shepherd Anthony C. Forster Maria Selmer 《Nucleic acids research》2013,41(20):9537-9548
RlmJ catalyzes the m6A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m6A MTases than to RNA m62A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer. 相似文献
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Molecular mechanisms of mammalian ribosome biogenesis remain largely unexplored. Here we develop a series of transposon-derived dominant mutants of Pes1, the mouse homolog of the zebrafish Pescadillo and yeast Nop7p implicated in ribosome biogenesis and cell proliferation control. Six Pes1 mutants selected by their ability to reversibly arrest the cell cycle also impair maturation of the 28S and 5.8S rRNAs in mouse cells. We show that Pes1 physically interacts with the nucleolar protein Bop1, and both proteins direct common pre-rRNA processing steps. Interaction with Bop1 is essential for the efficient incorporation of Pes1 into nucleolar preribosomal complexes. Pes1 mutants defective for the interaction with Bop1 lose the ability to affect rRNA maturation and the cell cycle. These data show that coordinated action of Pes1 and Bop1 is necessary for the biogenesis of 60S ribosomal subunits. 相似文献
11.
Leonhard Jakob Thomas Treiber Nora Treiber Alexander Gust Kevin Kramm Kerrin Hansen Mathias Stotz Ludwig Wankerl Franz Herzog Stefan Hannus Dina Grohmann Gunter Meister 《RNA (New York, N.Y.)》2016,22(3):383-396
In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing. 相似文献
12.
In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way. 相似文献
13.
Max Emperle Disha M Bangalore Sabrina Adam Stefan Kunert Hannah S Heil Katrin G Heinze Pavel Bashtrykov Ingrid Tessmer Albert Jeltsch 《Nucleic acids research》2021,49(14):8294
DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA. 相似文献
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The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor. 相似文献
15.
A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase beta (Pol beta) in mammalian base excision repair (BER). Pol beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity. 相似文献
16.
Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase 总被引:1,自引:0,他引:1
Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does. 相似文献
17.
Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III 总被引:1,自引:0,他引:1
Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members. 相似文献
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This review covers data on the structural organization of functional sites in the human ribosome, namely, the messenger RNA binding center, the binding site of the hepatitis C virus RNA internal ribosome entry site, and the peptidyl transferase center. The data summarized here have been obtained primarily by means of a site-directed cross-linking approach with application of the analogs of the respective ribosomal ligands bearing cross-linkers at the designed positions. These data are discussed taking into consideration available structural data on ribosomes from various kingdoms obtained with the use of cryo-electron microscopy, X-ray crystallography, and other approaches. 相似文献