Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A).

Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch.

Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS.

Molecular Anatomy of the Class I Ligase Ribozyme for Elucidation of the Activity-Generating Unit.

The class I ligase ribozyme consists of 121 nucleotides and shows a high catalytic rate comparable to that found in natural proteinaceous polymerases. In this study, we aimed to identify the smaller active unit of the class I ligase ribozyme comprising ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Based on the three-dimensional structure of the class I ligase ribozyme, mutants were prepared and their ligation activities were analyzed.

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them.

Peptide Bond Formation between Aminoacyl-Minihelices by a Scaffold Derived from the Peptidyl Transferase Center.

The peptidyl transferase center (PTC) in the ribosome is composed of two symmetrically arranged tRNA-like units that contribute to peptide bond formation. We prepared units of the PTC components with putative tRNA-like structure and attempted to obtain peptide bond formation between aminoacyl-minihelices (primordial tRNAs, the structures composed of a coaxial stack of the acceptor stem on the T-stem of tRNA). One of the components of the PTC, P1c2 (74-mer), formed a dimer and a peptide bond was formed between two aminoacyl-minihelices tethered by the dimeric P1c2.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNA with an extra guanosine residue at the 5'-terminus.

tRNA of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5'-terminus. However, the N. equitans tRNA mutant without extra guanosine at the 5'-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS).

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay.

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation.

Dissection of a rice OsMac1 mRNA 5' UTR to uncover regulatory elements that are responsible for its efficient translation.

The untranslated regions (UTRs) of mRNAs are involved in many posttranscriptional regulatory pathways. The rice OsMac1 mRNA has three splicing variants of the 5' UTR (UTRa, UTRb, and UTRc), which include a CU-rich region and three upstream open reading frames (uORFs). UTRc contains an additional 38-nt sequence, termed sp38, which acts as a strong translational enhancer of the downstream ORF; reporter analysis revealed translational efficiencies >15-fold higher with UTRc than with the other splice variants.

Correction to: G:U‑Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl‑tRNA Synthetase: An Insight into the Evolution of Aminoacyl‑tRNA Synthetases.

In the original version of this article, "A73" in Fig 6b was inadvertently labeled as "G73". The corrected Fig. 6 is given here.

Aminoacylation of short hairpin RNAs through kissing-loop interactions indicates evolutionary trend of RNA molecules.

The unique G3:U70 base pair in the acceptor stem of tRNA has been shown to be a critical recognition site by alanyl-tRNA synthetase (AlaRS). The base pair resides on one of the arms of the L-shaped structure of tRNA (minihelix) and the genetic code has likely evolved from a primordial tRNA-aaRS (aminoacyl-tRNA synthetase) system. In terms of the evolution of tRNA, incorporation of a G:U base pair in the structure would be important.

G:U-Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl-tRNA Synthetase: An Insight into the Evolution of Aminoacyl-tRNA Synthetases.

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.

Glycyl-tRNA synthetase from Nanoarchaeum equitans: The first crystal structure of archaeal GlyRS and analysis of its tRNA glycylation.

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.

Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation.

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants,  and , the influence of loop composition on ligation activity was investigated.

Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato.

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5'-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF.

DSH5, a dihydrosphingosine C4 hydroxylase gene family member, shows spatially restricted expression in rice and is lethal when expressed ectopically.

Dihydrosphingosine C4 hydroxylase (DSH), a diiron-binding membrane enzyme, catalyzes the hydration of dihydrosphingosine and acyl-sphinganine to produce phytosphingosine and phytoceramide, respectively. Rice has two types of DSH homologs: general DSHs, namely DSH1, DSH2 and DSH4, and others that show spatial expression profiles, namely DSH3 and DSH5. The general DSHs exist in many plant species.