Flexizymes: their evolutionary history and the origin of catalytic function

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.     

Reconsideration of the quantitative characterization of the reaction intermediate on electrocatalysts by a rotating ring-disk electrode: The intrinsic yield of H2O2 on Pt/C

To solve the problem of the catalyst-loading-effect on quantifying the reaction intermediates on the surface of electrocatalysts with a rotating ring-disk electrode, we studied the formation of hydrogen peroxide in the oxygen reduction reaction on Pt/C with various sample loadings and then proposed an extrapolation model for measuring the intrinsic yield of H₂O₂, which can quantitatively reflect the characteristics of the surface of a given catalyst. In the extrapolation model, the catalyst loading effect can be compensated by taking the catalyst loading-dependent probability of the re-adsorption + further reaction of the desorbed H₂O₂ into consideration. The core concept in this extrapolation model is that the probability of the re-adsorption + reaction of the desorbed H₂O₂ becomes zero if there is no other active site available (i.e., at the extrapolated hypothetical point of zero catalyst loading) for re-adsorption of the desorbed H₂O₂. The intrinsic yield of H₂O₂ by extrapolation was much higher than that measured by the conventional model, in which the re-adsorption + reaction of the desorbed H₂O₂ is not considered, and thus the catalyst loading-dependent apparent yield of H₂O₂ does not properly reflect the intrinsic characteristics of the surface of a given catalyst.     

Robust and accurate prediction of thermal error of machining centers under operations with cutting fluid supply

A novel temperature measuring system named LATSIS was proposed to realize a robust and accurate prediction of the thermal deformation of machining centers, even under external disturbances such as cutting fluid supply. LATSIS enables a drastic increase in the number of sensors employed for measuring the temperature of the machine tool. Thus, the entire temperature distribution can be obtained by interpolating the measured temperature 3-dimensionally without calculating the heat conduction. A set of experiments was conducted in which the LATSIS was employed to predict the TCP error. A total of 284 sensors were placed on the machining center, and the TCP error was predicted based on the measured temperature for the situation with/without the cutting fluid supply. The results of the prediction showed good agreement with the measured TCP error even during the initial transient temperature change as well as in the cooling phase after the machine halt. The TCP error with the cutting fluid supply is accurately predicted. LATSIS was proven to be a robust and accurate method for predicting the thermal deformation of machine tools, and is a promising technology for future manufacturing systems.     

One‐Pot/Four‐Step/Palladium‐Catalyzed Synthesis of Indole Derivatives: The Combination of Heterogeneous and Homogeneous Systems

One‐pot, four‐step syntheses of indoles using both solid‐supported heterogeneous and homogeneous palladium catalysts and reagents were carried out. Such a combination of these two‐phase catalysts and reagents causes a dramatic increase in yield, and it is a simple process. The presented methodology is effective for four‐step reactions to provide various functionalized indoles.     

Application of microbial genes to recalcitrant biomass utilization and environmental conservation

Recent papers concerning the application of microbial genes to recalcitrant biomass utilization and environmental conservation are reviewed. Microbial genes have been integrated and expressed in plants and microorganisms. When cellulose-degrading enzyme genes are expressed in rice plants, the transgenic plants exhibit swollen cell walls which increases the digestibility of rice straw in the rumen. When genes encoding aromatic compound-degrading enzymes are expressed in plants, it is expected that aromatic compounds contaminating soil would be degraded during the growth of the transgenic plants. The former transgenic plants are utilized as feed and the latter for phytoremediation. Dockerin and cohesin interactions occurring in the cellulase complex, cellulosome, are applied to the construction of artificial enzyme complexes and protein purification by expressing the genes in transformed bacteria and/or silkworms, respectively. In the case of the forced expression of bacterial genes encoding chitinase and/or hydrogenase in the wild-type bacteria, chitin degradation and hydrogen gas production in the transformed bacteria occur at much higher rates than in the wild type.     

XPS analysis of CdS/CuInSe2 heterojunctions

CdS/CuInSe₂ (CIS) heterojunctions were investigated by XPS analysis. An In-excess layer which may form an ordered vacancy compound (OVC) was present at the as-deposited CIS surface and it remained after chemical bath deposition of a CdS layer. The In-excess layer was removed by preferential etching with NH₃ aqueous solution. This result implies that the surface of the as-deposited CIS film was converted from the OVC with n-type conductivity into the CIS with p-type by NH₃ treatment. The conduction band offsets at the CdS/p-CIS and CdS/n-OVC were determined to be 1.0 and 0.3 eV, respectively. The CIS solar cells fabricated with n-OVC surface layer exhibited higher cell efficiencies than those fabricated with p-CIS surface layer.     

Evaluating the performance of forced convection heat transfer enhancement

Two methods for assessing thermal performance were evaluated for four kinds of forced convective heat transfer augmentations. On method uses the first law of thermodynamics, i.e., the heat transfer improvement at (1) constant Reynolds number, (2) constant pressure loss, and (3) constant pumping power. The other method uses the second law of thermodynamics, i.e., the entropy generation. The first method restricts the effective region and the second method supplies the condition for achieving the minimum entropy generation rate. © 1998 Scripta Technica, Heat Trans Jpn Res, 27(2): 142–154, 1998     

Effects of CdS buffer layers on photoluminescence properties of Cu(In,Ga)Se2 solar cells

Photoluminescence (PL) have been studied on Cu(In,Ga)Se₂ (CIGS) thin films, CdS/CIGS and CIGS solar cells, to clarify the carrier recombination process. The chemical-bath deposition (CBD) of the CdS buffer layer on the CIGS thin film leads to (i) the enhancement of near-band-edge PL intensity by a factor of 2–3, (ii) change in energy of the defect-related PL and (iii) the slight change in the decay time. They are related not only to the minimization of the surface recombination but also to the modification of native defects at the Cu-poor surface of CIGS by the occupation of Cd atom at the Cu site. A donor–acceptor pair PL at low-temperature and temperature-dependent PL have been studied. They are discussed in terms of the impurity and defect levels created in the CIGS film during the CBD-CdS process.     

Composition of gaseous combustion products of polymers

The gaseous products generated by the flaming combustion of ten kinds of synthetic polymers and a kind of wood (cedar) under the same conditions (sample weight, 0.1 g; temperature, 700°C air flow rates, 50 and 100 l./hr) were quantitatively analyzed by infrared spectrophotometry, gas chromatography, and colorimetric tube method. The main hydrocarbons generated were methane, ethylene, and acetylene. The amount of acetylene generated by the flaming combustion of polymers was much larger than the amount of acetylene formed by pyrolysis at 700°C in nitrogen. Acetylene increased in quantity with increasing air. For nitrogen compounds, hydrogen cyanide was generated from every polymer containing nitrogen used, but ammonia was detected only for nylon 66 and polyacrylamide. Nitrogen monoxide and nitrogen dioxide were detected only in small amounts. Nitrous oxide was detected in the gaseous products generated by the nonflaming combustion of urea resin and melamin resin. It was also found that about 70% of the nitrogen in N-66 and PAA was converted into nitrogen gas (N₂) by combustion under the conditions described above.