ArcS from Thermococcus kodakarensis transfers L-lysine to preQ nucleoside derivatives as minimum substrate RNAs.

Archaeosine (G) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ). In Euryarchaea, preQ15 in tRNA is further modified by archaeosine synthase (ArcS).

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET).

Functional analysis of tRNA modification enzymes using mutational profiling.

Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme.

Escherichia coli tRNA (Gm18) methyltransferase (TrmH) requires the correct localization of its methylation site (G18) in the D-loop for efficient methylation.

TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E.

Transfer RNA Modification Enzymes with a Thiouridine Synthetase, Methyltransferase and Pseudouridine Synthase (THUMP) Domain and the Nucleosides They Produce in tRNA.

The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA mA22 methyltransferase.

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure.

A selective and sensitive detection system for 4-thiouridine modification in RNA.

4-Thiouridine (sU) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of sU in tRNA are ongoing in the tRNA modification field. sU has also recently been utilized as a biotechnological tool for analysis of RNAs.

Genetic and Functional Analyses of Archaeal ATP-Dependent RNA Ligase in C/D Box sRNA Circularization and Ribosomal RNA Processing.

RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates.

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine--) Methyltransferase (Trm11-Trm112 Complex).

The Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms -methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112.

Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs.

The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed.

Identification of a radical SAM enzyme involved in the synthesis of archaeosine.

Archaeosine (G), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ)-containing tRNA (qN-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G-containing tRNA (G-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to qN-tRNA to form qkN-tRNA, which has a preQ lysine adduct as a base.

Structure of tRNA methyltransferase complex of Trm7 and Trm734 reveals a novel binding interface for tRNA recognition.

The complex between Trm7 and Trm734 (Trm7-Trm734) from Saccharomyces cerevisiae catalyzes 2'-O-methylation at position 34 in tRNA. We report biochemical and structural studies of the Trm7-Trm734 complex. Purified recombinant Trm7-Trm734 preferentially methylates tRNAPhe transcript variants possessing two of three factors (Cm32, m1G37 and pyrimidine34).

Distinct Modified Nucleosides in tRNA from the Hyperthermophilic Archaeon Thermococcus kodakarensis and Requirement of tRNA mG10/mG10 Methyltransferase (Archaeal Trm11) for Survival at High Temperatures.

tRNA mG10/mG10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms ,-dimethylguanosine (mG10) via -methylguanosine (mG10). We determined the complete sequence of tRNA, one of the substrate tRNAs for archaeal Trm11 from , a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNA identified 15 types of modified nucleoside at 21 positions.

Regulatory Factors for tRNA Modifications in Extreme- Thermophilic Bacterium .

is an extreme-thermophilic bacterium that can grow at a wide range of temperatures (50-83°C). To enable to grow at high temperatures, several biomolecules including tRNA and tRNA modification enzymes show extreme heat-resistance. Therefore, the modified nucleosides in tRNA from have been studied mainly from the view point of tRNA stabilization at high temperatures.

Challenges and opportunities for eliminating tuberculosis - leveraging political momentum of the UN high-level meeting on tuberculosis.

Background: As demonstrated by the United Nations High-Level Meeting on tuberculosis (TB) held in September 2018, the political momentum for TB has been increasing. The aim of this study was to analyze the current challenges and opportunities for global TB control and, with specific focus on policies surrounding TB control, to reveal what kinds of efforts are needed to accelerate global TB control.

Methods: We organized two expert meetings with the purposes of assessing the current situation and analyzing challenges regarding TB control.

Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA.

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted.

Consumption of N5, N10-methylenetetrahydrofolate in Thermus thermophilus under nutrient-poor condition.

TrmFO catalyzes the formation of 5-methyluridine at position 54 in tRNA and uses N5, N10-methylenetetrahydrofolate (CH2THF) as the methyl group donor. We found that the trmFO gene-disruptant strain of Thermus thermophilus, an extremely thermophilic eubacterium, can grow faster than the wild-type strain in the synthetic medium at 70°C (optimal growth temperature). Nucleoside analysis revealed that the majority of modifications were appropriately introduced into tRNA, showing that the limited nutrients are preferentially consumed in the tRNA modification systems.

The RNA-splicing endonuclease from the euryarchaeaon Methanopyrus kandleri is a heterotetramer with constrained substrate specificity.

Four different types (α4, α'2, (αβ)2 and ϵ2) of RNA-splicing endonucleases (EndAs) for RNA processing are known to exist in the Archaea. Only the (αβ)2 and ϵ2 types can cleave non-canonical introns in precursor (pre)-tRNA. Both enzyme types possess an insert associated with a specific loop, allowing broad substrate specificity in the catalytic α units.

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene.

Kinetic characterization of substrate-binding sites of thermostable tRNA methyltransferase (TrmB).

TrmB is a eubacterial tRNA methyltransferase which catalyzes the formation of N7-methylguanosine at position 46 (m7G46) in tRNA consuming S-adenosyl-L-methionine (AdoMet) as the methyl group donor during the reaction. Previously, we purified TrmB from Aquifex aeolicus, a hyper-thermophilic eubacterium, and clarified the recognition sites in tRNA. Furthermore, we reported that an additional C-terminal region of A.

Long and branched polyamines are required for maintenance of the ribosome, tRNA and tRNA in Thermus thermophilus cells at high temperatures.

Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T.

Transfer RNA methyltransferases with a SpoU-TrmD (SPOUT) fold and their modified nucleosides in tRNA.

The existence of SpoU-TrmD (SPOUT) RNA methyltransferase superfamily was first predicted by bioinformatics. SpoU is the previous name of TrmH, which catalyzes the 2'-Omethylation of ribose of G18 in tRNA; TrmD catalyzes the formation of N1-methylguanosine at position 37 in tRNA. Although SpoU (TrmH) and TrmD were originally considered to be unrelated, the bioinformatics study suggested that they might share a common evolution origin and form a single superfamily.

Structural and functional analyses of the archaeal tRNA m2G/m22G10 methyltransferase aTrm11 provide mechanistic insights into site specificity of a tRNA methyltransferase that contains common RNA-binding modules.

N(2)-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N(2)-methylguanosine and N(2),N(2)-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM.

Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures.

TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T.