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tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5ʹ-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5ʹ-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 A resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5ʹ-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

The nucleosome is the basic organizational unit of the genome. The folding structure of nucleosomes is closely related to genome functions, and has been reported to be in dynamic interplay with binding of various nuclear proteins to genomic loci. Here, we describe our high-throughput chromosome conformation capture with nucleosome orientation (Hi-CO) technology to derive 3D nucleosome positions with their orientations at every genomic locus in the nucleus. This technology consists of an experimental procedure for nucleosome proximity analysis and a computational procedure for 3D modeling. The experimental procedure is based on an improved method of high-throughput chromosome conformation capture (Hi-C) analysis. Whereas conventional Hi-C allows spatial proximity analysis among genomic loci with 1-10 kbp resolution, our Hi-CO allows proximity analysis among DNA entry or exit points at every nucleosome locus. This analysis is realized by carrying out ligations among the entry/exit points in every nucleosome in a micrococcal-nuclease-fragmented genome, and by quantifying frequencies of ligation products with next-generation sequencing. Our protocol has enabled this analysis by cleanly excluding unwanted non-ligation products that are abundant owing to the frequent genome fragmentation by micrococcal nuclease. The computational procedure is based on simulated annealing-molecular dynamics, which allows determination of optimized 3D positions and orientations of every nucleosome that satisfies the proximity ligation data sufficiently well. Typically, examination of the Saccharomyces cerevisiae genome with 130 million sequencing reads facilitates analysis of a total of 66,360 nucleosome loci with 6.8 nm resolution. The technique requires 2-3 weeks for sequencing library preparation and 2 weeks for simulation.

Nanoarchaeum equitans is a species of hyperthermophilic archaea with the smallest genome size. Its alanyl-tRNA synthetase genes are split into AlaRS-α and AlaRS-β, encoding the respective subunits. In the current report, we surveyed N. equitans AlaRS-dependent alanylation of RNA minihelices, composed only of the acceptor stem and the T-arm of tRNAAla. Combination of AlaRS-α and AlaRS-β showed a strong alanylation activity specific to a single G3:U70 base pair, known to mark a specific tRNA for charging with alanine. However, AlaRS-α alone had a weak but appreciable alanylation activity that was independent of the G3:U70 base pair. The shorter 16-mer RNA tetraloop substrate mimicking only the first four base pairs of the acceptor stem of tRNAAla, but with C3:G70 base pair, was also successfully aminoacylated by AlaRS-α. The end of the acceptor stem, including CCA-3' terminus and the discriminator A73, was able to function as a minimal structure for the recognition by the enzyme. Our findings imply that aminoacylation by N. equitans AlaRS-α may represent a vestige of a primitive aminoacylation system, before the appearance of the G3:U70 pair as an identity element for alanine.

This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans — a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long β-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.

The origin of homochirality in L-amino acid in proteins is one of the mysteries of the evolution of life. Experimental studies show that a non-enzymatic aminoacylation reaction of an RNA minihelix has a preference for L-amino acid over D-amino acid. The reaction initiates by approaching of a 3′-oxygen of the RNA minihelix to the carbonyl carbon of an aminoacyl phosphate oligonucleotide. Here, employing molecular dynamics simulations, we examined the possible mechanisms that determine this chiral selectivity. The simulation system adopted a geometry required for the chemical reaction to occur more frequently with L-alanine than that with D-alanine. For L-alanine, the structure with this geometry was formed by a combination of stable dihedral angles along alanyl phosphate backbone with a canonical RNA structure, where the methyl group of alanine was placed on the opposite side of the approaching 3′-hydroxyl group with respect to the carbonyl plane. For D-alanine, the methyl group and the 3′-hydroxyl group were placed on the same side with respect to the carbonyl plane, which significantly decreased its ability to approach 3′-oxygen close to the carbonyl carbon compared to L-alanine. The mechanism suggested herein can explain experimentally observed chiral preferences.

Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90° left-handedly, thereby generating directional LR asymmetry. We recently revealed that the hindgut epithelial cell is chiral in shape and other properties; this is termed planar cell chirality (PCC). We previously showed by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. Here, to understand how PCC directs the asymmetric cell-shape, we analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Our mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, our results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance.

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology. DOI: http://dx.doi.org/10.7554/eLife.19274.001, eLife digest Much of the work that has been done to understand how cells work has involved studying parts of a cell in isolation. This is particularly true of studies that have examined the arrangement of atoms in large molecules with elaborate structures like proteins or DNA. However, cells are densely packed with many different molecules and there is little proof that proteins keep the same structures inside cells that they have when they are studied alone. To really understand how cells work, new ways to understand how molecules behave inside cells are needed. While this cannot be achieved directly, technology has now reached the stage where we can, to some extent, study living cells by recreating them virtually. Simulated cells can copy the atomic details of all the molecules in a cell and can estimate how different molecules might behave together. Yu et al. have now developed a computer simulation of part of a cell from the bacterium, Mycoplasma genitalium, one of the simplest forms of life on Earth. This model suggested new possible interactions between molecules inside cells that cannot currently be studied in real cells. The model shows that some proteins have a much less rigid structure in cells than they do in isolation, whilst others are able to work together more closely to carry out certain tasks. Finally, the model predicted that small molecules such as food, water and drugs would move more slowly through cells as they become stuck or trapped by larger molecules. These results could be particularly important in helping to improve drug design. Currently the simulations are limited, and can only model parts of simple cells for less than a thousandth of a second. However, in future it should be possible to recreate larger and more complex cells, including human cells, for longer periods of time. These could be used to better study human diseases and help to design new treatments. The ultimate goal is to simulate a whole cell in full detail by combining all the available experimental data. DOI: http://dx.doi.org/10.7554/eLife.19274.002

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and β-rhombus analogous to α helix and β sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

The effect of macromolecular crowding on protein diffusion has been extensively studied in order to understand the protein dynamics in cellular environments. Previous studies have generally focused on proteins as crowding agents as they are known to occupy 20-30% of the cell volume. The volume fraction of the nucleoid in bacterial cells is also significant, around 10-20%, but the effect of chromosomal DNA as a crowding agent on protein diffusion has drawn relatively limited attention. Using recently build high-resolution models of bacterial nucleoids, Brownian and Stokesian dynamics simulations of coarse-grained model systems containing chromosomal DNA and proteins were carried out to investigate the effect of nucleoid crowding on protein diffusion. The coarse-grained model for the nucleoid is an experimentally-derived 3D model of the Caulobacter crescentus chromosome at base-pair resolution built by combining topological information of chromosomal DNA with distance restraints obtained from high-throughput Chromosome Conformation Capture (HiC) experiments, while proteins in the systems are modeled as hard-spheres. Results show that diffusion of proteins are slower around the nucleoid compared to in protein crowding and that there is size-dependent exclusion of proteins from the chromosome interior. Further analysis was carried to examine the degree of anomalous diffusion at different time and length scales.

Chromatin dynamics and arrangement are involved in many biological processes in nuclei of eukaryotes including plants. Plants have to respond rapidly to various environmental stimuli to achieve growth and development because they cannot move. It is assumed that the alteration of chromatin dynamics and arrangement support the response to these stimuli; however, there is little information in plants. In this study, we investigated the chromatin dynamics and arrangement with DNA damage in Arabidopsis thaliana by live-cell imaging with the lacO/LacI-EGFP system and simulation analysis. It was revealed that homologous loci kept a constant distance in nuclei of A. thaliana roots in general growth. We also found that DNA double-strand breaks (DSBs) induce the approach of the homologous loci with γ-irradiation. Furthermore, AtRAD54, which performs an important role in the homologous recombination repair pathway, was involved in the pairing of homologous loci with γ-irradiation. These results suggest that homologous loci approach each other to repair DSBs and AtRAD54 mediates these phenomena.

DNA binding proteins efficiently search for their cognitive sites on long genomic DNA by combining 3D diffusion and 1D diffusion (sliding) along the DNA. Recent experimental results and theoretical analyses revealed that the proteins show a rotation-coupled sliding along DNA helical pitch. Here, we performed Brownian dynamics simulations using newly developed coarse-grained protein and DNA models for evaluating how hydrodynamic interactions between the protein and DNA molecules, binding affinity of the protein to DNA, and DNA fluctuations affect the one dimensional diffusion of the protein on the DNA. Our results indicate that intermolecular hydrodynamic interactions reduce 1D diffusivity by 30%. On the other hand, structural fluctuations of DNA give rise to steric collisions between the CG-proteins and DNA, resulting in faster 1D sliding of the protein. Proteins with low binding affinities consistent with experimental estimates of non-specific DNA binding show hopping along the CG-DNA. This hopping significantly increases sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome., Author Summary DNA binding proteins efficiently search for their cognitive sites on long genomic DNA in cells to control biological activities. Recent experimental studies have revealed that the proteins use not only three-dimensional diffusion, but also one-dimensional diffusion (sliding) on DNA for this search process. For a better understanding of this biological process, we need to elucidate the mechanism of sliding. We report here molecular simulations using newly developed coarse-grained protein and DNA models for elucidating the nature of the sliding motions. Our simulation results show that: 1) hydrodynamic interactions between protein and DNA reduce sliding rate by 30%, 2) structural fluctuations of DNA give rise to steric collisions between proteins and DNA, which facilitate sliding motions, and 3) proteins with low binding affinities to DNA can hop along the DNA, resulting in a significant increase in sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome.

Structural genomics projects are beginning to produce protein structures with unknown functions, thereby creating a need for high-throughput methods to predict functions. Although sequence-based function prediction methods have been used extensively, structure-based prediction is believed to provide higher specificity and sensitivity because functions are closely related to the three-dimensional structures of functional sites, which are more strongly conserved during evolution than sequence. We have developed FCANAL, a method to predict functions using a score matrix obtained from the distances between Cα atoms and frequencies of appearance [1]. The previous report used key residues predicted from sequence comparisons (motifs). In this report, we have expanded the method to include enzymes and binding proteins with key residues predicted on the basis of three-dimensional structures. Using FCANAL, we constructed score matrices for 31 enzymes. When we applied them to all of the structure entries deposited in the Protein Data Bank, FCANAL could detect functional sites with high accuracy. This suggests that FCANAL will help identify the functions of newly determined structures and pinpoint their functionally important regions.

Some organs in animals display left-right (LR) asymmetry. To better understand LR asymmetric morphogenesis in Drosophila, we studied LR directional rotation of the hindgut epithelial tube. Hindgut epithelial cells adopt a LR asymmetric (chiral) cell shape within their plane, and we refer to this cell behavior as planar cell-shape chirality (PCC). Drosophila E-cadherin (DE-Cad) is distributed to cell boundaries with LR asymmetry, which is responsible for the PCC formation. Myosin ID switches the LR polarity found in PCC and in DE-Cad distribution, which coincides with the direction of rotation. An in silico simulation showed that PCC is sufficient to induce the directional rotation of this tissue. Thus, the intrinsic chirality of epithelial cells in vivo is an underlying mechanism for LR asymmetric tissue morphogenesis.

WL-O系統,WL-B系統及び内在性ウイルス遺伝子の部分発現のある個体群の3群を用いて,内在性ウイルス遺伝子と細胞表面レセプター遺伝子が鶏染色体上でどういう位置関係にあるのかを後代検定法を用いて調査した。その結果,gs抗原とchf活性の分離比はそれぞれの遺伝子座が同一染色体上にある場合の期待値に近く,我々は,少なくとも一対のgag遺伝子とenv遺伝子が同一染色体上にあるものと結論した。そして,これらの遺伝子は,ev-3遺伝子座のGsとgsの対立遺伝子と同じ性質のものであろうと推定した。一方,B亜群ウイルスに対する細胞の感受性と,gs抗原及びchf活性の分離比については,両座位が独立の場合の期待分離比に近いので,B亜群ウイルスに対する細胞のレセプター遺伝子座とこの内在性ウイルス遺伝子座とは異なる染色体上にあるものと思われた。

鶏白血病ウイルス(ALV)抗体測定法としての酵素抗体法(ELISA)について抗原抗体反応系の解析を行い,さらに血清中和法(SN),蛍光抗体法(FA)との相関関係と感度の比較を行った。1)A亜群,B亜群2種の抗原に対する抗体価の推移をELISA,SN,FAで確認すると,ELISA,FAでは主として群特異抗体(gs抗体)を,SNは亜群特異抗体を検出していると思われた。2)gs抗体を産生する抗原成分として3種の蛋白が確認された。そして,それぞれに対する抗体の発現順序は,まずp27,つぎにgp85,さらにp15に対する抗体であった。3)1野外鶏群について,3種の測定方法で算出した抗体価の関係を見ると,いずれにも相関が認められた。4)測定方法による検出感度を比較すると,ELISAは,SNの約10倍,FAはSNとほぼ同感度であった。

In order to demonstrate that the extracorporeal shockwave lithotripsy (ESWL) for urolithiasis causes a renal damage, we immunoassayed S100a0 protein and creatine kinase isozymes (CK-B & CK-M) in the serum and urine from patients with renal stone (N = 21) and those with ureteral stone (N = 18) before and after ESWL. S100a0 protein and CK-B, which are localized in the proximal renal tubules at relatively high concentrations, were enhanced more or less both in the urine and serum samples after the treatment in both groups of patients. Since CK-M, which is mainly localized in muscle tissue, also increased in both groups of patients, S100a0 protein and CK-B may be derived not only from kidney, but also from muscle tissues.

The presence of chick helper factor (chf) in chick embryo fibroblasts (CEF) which lack the natural group-specific (gs) antigen of avian leukosis was determined in a simple Mendelian manner from the dominant autosomal gene. The presence or absence of chf determined the susceptibility to subgroup E virus in the WL-O line, but not in the WL-B line, which had the fixed C/BE character. Dimethyl sulfoxide (DMSO) together with BH-RSV(RAV-7) promoted the appearance of chf in gs − chf − CEF after long-term culture.