In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

N⁶-threonylcarbamoyladenosine (t⁶A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t⁶A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t⁶A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t⁶A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t⁶A in nature. These findings shed light on the reaction mechanism of t⁶A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells.     

Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification

N⁶-threonylcarbamoyladenosine (t⁶A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t⁶A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t⁶A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t⁶A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.     

Qri7/OSGEPL,the mitochondrial version of the universal Kae1/YgjD protein,is essential for mitochondrial genome maintenance

Yeast Qri7 and human OSGEPL are members of the orthologous Kae1(OSGEP)/YgjD protein family, the last class of universally conserved proteins without assigned function. Phylogenetic analyses indicate that the eukaryotic Qri7(OSGEPL) proteins originated from bacterial YgjD proteins. We have recently shown that the archaeal Kae1 protein is a DNA-binding protein that exhibits apurinic endonuclease activity in vitro. We show here that the Qri7/OSGEPL proteins localize in mitochondria and are involved in mitochondrial genome maintenance in two model eukaryotic organisms, Saccharomyces cerevisiae and Caenorhabditis elegans. Furthermore, S. cerevisiae Qri7 complements the loss of the bacterial YgjD protein in Escherichia coli, suggesting that Qri7/OSGEPL and YgjD proteins have retained similar functions in modern organisms. We suggest to name members of the Kae1(OSGEP)/YgjD family UGMP, for Universal Genome Maintenance Proteins.     

Structure of hydrogenase maturation protein HypF with reaction intermediates shows two active sites

[NiFe]-hydrogenases are multimeric proteins. The?large subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands is?catalyzed by the combined actions of HypF and?HypE using carbamoylphosphate as a substrate.?We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ～14??. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.     

Anti-Japanese-encephalitis-viral effects of kaempferol and daidzin and their RNA-binding characteristics

Background

New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an α-1 translational frameshift to synthesize the NS1'' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described.

Methodology/Principal Findings

The 50% cytotoxic concentration (CC₅₀) and 50% effective concentration (EC₅₀) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC₅₀ values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC₅₀ was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent K_b value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could.

Conclusions/Significance

Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.     

A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex -- the KEOPS complex -- containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 cgi121 mutants. Deletion of BUD32 or other KEOPS components leads to short telomeres and a failure to add telomeres de novo to DNA double-strand breaks. Our results therefore indicate that the KEOPS complex promotes both telomere uncapping and telomere elongation.     

The structure and function of MCM from archaeal M. Thermoautotrophicum

Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.     

Evidence that Pituitary Cation-Sensitive Neutral Endopeptidase Is a Multicatalytic Protease Complex

Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.     

Activation of human meiosis-specific recombinase Dmc1 by Ca2+

Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.     

The Nijmegen breakage syndrome gene and its role in genome stability

NBS1 is the key regulator of the RAD50/MRE11/NBS1 (R/M/N) protein complex, a sensor and mediator for cellular DNA damage response. NBS1 potentiates the enzymatic activity of MRE11 and directs the R/M/N complex to sites of DNA damage, where it forms nuclear foci by interacting with phosphorylated H2AX. The R/M/N complex also activates the ATM kinase, which is a major kinase involved in the activation of DNA damage signal pathways. The ATM requires the R/M/N complex for its own activation following DNA damage, and for conformational change to develop a high affinity for target proteins. In addition, association of NBS1 with PML, the promyelocytic leukemia protein, is required to form nuclear bodies, which have various functions depending on their location and composition. These nuclear bodies function not only in response to DNA damage, but are also involved in telomere maintenance when they are located on telomeres. In this review, we describe the role of NBS1 in the maintenance of genetic stability through the activation of cell-cycle checkpoints, DNA repair, and protein relocation.     

The structural and functional workings of KEOPS

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway–Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N⁶-threonylcarbamoyl adenosine (t⁶A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.