Molecular mechanism of divalent-metal-induced activation of NS3 helicase and insights into Zika virus inhibitor design
Zika virus has attracted increasing attention because of its potential for causing human neural disorders, including microcephaly in infants and Guillain–Barré syndrome. Its NS3 helicase domain plays critical roles in NTP-dependent RNA unwinding and translocation during viral replication. Our structural analysis revealed a pre-activation state of NS3 helicase in complex with GTPS, in which the triphosphate adopts a compact conformation in the absence of any divalent metal ions. In contrast, in the presence of a divalent cation, GTPS adopts an extended conformation, and the Walker A motif undergoes substantial ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Cao, X., Li, Y., Jin, X., Li, Y., Guo, F., Jin, T. Tags: Structural Biology Source Type: research
Structure of the 70S ribosome from human pathogen Staphylococcus aureus
Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Khusainov, I., Vicens, Q., Bochler, A., Grosse, F., Myasnikov, A., Menetret, J.-F., Chicher, J., Marzi, S., Romby, P., Yusupova, G., Yusupov, M., Hashem, Y. Tags: Structural Biology Source Type: research
Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer
Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-met...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Lin, J. L. J., Wu, C.-C., Yang, W.-Z., Yuan, H. S. Tags: Structural Biology Source Type: research
Dynamic peptides of human TPP1 fulfill diverse functions in telomere maintenance
Telomeres are specialized nucleoprotein complexes that comprise the ends of linear chromosomes. Human telomeres end in a short, single-stranded DNA (ssDNA) overhang that is recognized and bound by two telomere proteins, POT1 and TPP1. Whereas POT1 binds directly to telomere ssDNA, its interaction with TPP1 is essential for localization of POT1 to the telomere. TPP1 also provides enhanced binding and sequence discrimination that regulates POT1-TPP1 interactions exclusively with telomere ssDNA. Finally, TPP1 recruits telomerase, the enzyme responsible for synthesis of telomere DNA, to the telomere. While the oligosaccharide&...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Rajavel, M., Orban, T., Xu, M., Hernandez-Sanchez, W., de la Fuente, M., Palczewski, K., Taylor, D. J. Tags: Structural Biology Source Type: research
Cytoplasmic Drosha activity generated by alternative splicing
RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Alth...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Dai, L., Chen, K., Youngren, B., Kulina, J., Yang, A., Guo, Z., Li, J., Yu, P., Gu, S. Tags: RNA Source Type: research
Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs
The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here, we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of maturing snRNAs during their cytoplasmic processing. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exo...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Łabno, A., Warkocki, Z., Kulinski, T., Krawczyk, P. S., Bijata, K., Tomecki, R., Dziembowski, A. Tags: RNA Source Type: research
Molecular basis of RNA guanine-7 methyltransferase (RNMT) activation by RAM
Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Varshney, D., Petit, A.-P., Bueren-Calabuig, J. A., Jansen, C., Fletcher, D. A., Peggie, M., Weidlich, S., Scullion, P., Pisliakov, A. V., Cowling, V. H. Tags: RNA Source Type: research
The target spectrum of SdsR small RNA in Salmonella
Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these ‘core sRNAs’ of largely unknown function is the abundant ~100-nucleotide SdsR sRNA which is transcribed by the general stress -factor, S and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present i...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Fröhlich, K. S., Haneke, K., Papenfort, K., Vogel, J. Tags: RNA Source Type: research
A highly specific sodium aptamer probed by 2-aminopurine for robust Na+ sensing
Sodium is one of the most abundant metals in the environment and in biology, playing critical ecological and physiological roles. Na+ is also the most common buffer salt for nucleic acids research, while its specific interaction with DNA has yet to be fully studied. Herein, we probe a highly selective and robust Na+ aptamer using 2-aminopurine (2AP), a fluorescent adenine analog. This aptamer has two DNA strands derived from the Ce13d DNAzyme. By introducing a 2AP at the cleavage site of the substrate strand, Na+ induces ~40% fluorescence increase. The signaling is improved by a series of rational mutations, reaching >6...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Zhou, W., Ding, J., Liu, J. Tags: Nucleic Acid Enzymes Source Type: research
Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems
Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei. Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Beguin, P., Charpin, N., Koonin, E. V., Forterre, P., Krupovic, M. Tags: Nucleic Acid Enzymes Source Type: research
DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism
The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Gubaev, A., Weidlich, D., Klostermeier, D. Tags: Nucleic Acid Enzymes Source Type: research
Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells
We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a v...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Artusi, S., Perrone, R., Lago, S., Raffa, P., Di Iorio, E., Palu, G., Richter, S. N. Tags: Molecular Biology Source Type: research
Mechanistic determinants of MBNL activity
Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUGexp) RNAs. Pathogenic interactions between MBNL and CUGexp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To addr...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Sznajder, Łukasz J., Michalak, M., Taylor, K., Cywoniuk, P., Kabza, M., Wojtkowiak-Szlachcic, A., Matłoka, M., Konieczny, P., Sobczak, K. Tags: Molecular Biology Source Type: research
The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter
Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results i...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Łyzen, R., Maitra, A., Milewska, K., Kochanowska-Łyzen, M., Hernandez, V. J., Szalewska-Pałasz, A. Tags: Molecular Biology Source Type: research
Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA
Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP ...
Source: Nucleic Acids Research - December 1, 2016 Category: Research Authors: Robb, N. C., te Velthuis, A. J. W., Wieneke, R., Tampe, R., Cordes, T., Fodor, E., Kapanidis, A. N. Tags: Molecular Biology Source Type: research
