Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: YqeK is a Ap 4 A hydrolase. A , analytical size-exclusion chromatography of purified Bs YqeK and Gt YqeK. Both proteins elute at approximately 42 kDa, consistent with a homodimer. The grey shaded profile and calibration line represent the molecular-weight standards used to estimate the apparent molecular masses. The inset shows the SDS-PAGE of Bs YqeK and Gt YqeK. B , HPLC analysis of Ap 4 A hydrolysis by Bs YqeK with or without manganese, which promotes symmetrically converts of Ap 4 A into ADP. C , ITC analysis of Ap 4 A binding to B. subtilis YqeK. D , enzyme-kinetic analysis of B. subtilis YqeK mediated hydrolysis of Ap 4 A. A hyperbolic fit according to Michaelis-Menten was used to determine K m and V max . E , 5′-Ap 4 A-RNA decapping of Bs YqeK in vitro. Schematic representation of the 5′-Ap 4 A-RNA decapping reaction ( left ) and analysis of in vitro decapping ( right ) of WT Bs YqeK and Bs YqeK (H50A/D51A) mutant proteins and the positive control Ec ApaH using APB urea polyacrylamide gel electrophoresis.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Size-exclusion Chromatography, Purification, Molecular Weight, SDS Page, Analysis, Binding Assay, In Vitro, Mutagenesis, Positive Control, Polyacrylamide Gel Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Relative Activity and Binding Affinity of YqeK Toward Ap 4 N Substrates

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Activity Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Quantification of intracellular Ap 4 N dinucleotides. Targeted QQQ LC–MS/MS analysis of Ap 4 A, Ap 4 G, Ap 4 C and Ap 4 U in B. subtilis WT 168, deletion (Δ yqeK ), overexpression (+ yqeK ) and complementation (Δ yqeK + yqeK ) strains at OD 600 0.5 ( A ), 1.0 ( B ), 3.0 ( C ) and overnight culture, OD 600 2.0 ( D ). Metabolite levels were normalized to dilution-corrected culture density and are shown as pmol/OD 600 .ml. Bars indicate mean values; points represent nine measurements from three biological replicates, with three technical replicates per clone. Statistical analyses were performed using GraphPad Prism. Technical triplicates were averaged within each biological replicate, and each growth stage was analyzed separately by two-way ANOVA followed by Dunnett’s multiple-comparisons test against Δ yqeK within each nucleotide group. Adjusted p values are indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Liquid Chromatography with Mass Spectroscopy, Analysis, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Structural insights into Gt YqeK. Overall structures of Gt YqeK ( A ) in the apo state, ( B ) in a complex with ADP, and ( C ) in a complex with the non-hydrolysable Ap 4 A analog Ap 2 Cp 2 A. ( D ) Detailed view of the deeply buried nucleotide-binding pocket in Gt YqeK. E, F , structural comparison of the ADP- and Ap 2 Cp 2 A-bound states reveal rearrangements of individual amino acid side chains in the phosphate-binding region and the second nucleotide-binding pocket.

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Binding Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Molecular insights into the promiscuous Ap 4 N hydrolase YqeK

doi: 10.1016/j.jbc.2026.113490

Figure Lengend Snippet: Ap 4 A Hydrolysis and Binding Affinity by YqeK Variants

Article Snippet: The reaction was performed using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S) with the addition of 8 mM Ap 4 A (Jena Bioscience), 10 mM UTP and CTP, and 2 mM GTP.

Techniques: Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Recoding by N 1 -methylpseudouridine guides rational mRNA vaccine design

doi: 10.1038/s41467-026-77796-3

Figure Lengend Snippet: a Non-slippery synonymous UUCC (or m 1 Ψm 1 ΨCC) sequence prevents base-pairing with tRNA Phe (blue) in the +1 frame. b Synthesis of 0-frame fMAFR (open circles and dotted line) and +1-frame fMAFV (closed circles and solid line) products during translation of UUCC (gray) and m 1 Ψm 1 ΨCC (maroon) at decreasing Arg-tRNA Arg to ribosome ratio. Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Statistical significance by ANOVA test: *** P = 0.0008 (F(1, 4) = 84.08; df = 1); ** P = 0.0020 (F(1, 4) = 50.86; df = 1). c Schematic of the dual-fluorescence mRNA reporter containing synonymous, non-slippery UUCC substitution. d Representative sfGFP/mChy ratio distribution after transfection of HEK293T with mRNA containing only mChy (light blue for unmodified; dark blue for m 1 Ψ-modified), sfGFP in-frame with mChy ( mChy-sfGFP ; light orange for unmodified and dark orange for m 1 Ψ-modified), and mRNA reporters containing the BNT162b2 U 1543 U 1544 C 1545 C 1546 non-slippery variant (gray for unmodified; maroon for m 1 Ψ-modified). e +1 frameshifting on the synonymous non-slippery U 1543 U 1544 C 1545 C 1546 variant after transfection with unmodified (gray) or m 1 Ψ-modified (maroon) mRNA reporters. Shown are values in individual replicates (black circles) and mean values with error bars representing SE from n = 3 biological replicates. ns, P = 0.5012 ( t = 0.7573; df = 3.170), according to two-tailed Welch’s t -test. f Schematic of the dual-fluorescence mRNA reporter carrying nucleotide substitutions (gray) downstream of the slippery codon U 1543 U 1544 U 1545 (purple). g Change in +1 frameshifting on m 1 Ψ-modified, compared to unmodified, mRNA reporters in HEK293T cells, as compared to the dwell time of the ribosome on the respective A-site codon . Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Dashed line shows the mean fold change in +1 frameshifting for codons with first-position C (black) and U (gray). h Mean fold change in +1 frameshifting in m 1 Ψ-modified compared to unmodified mRNA for a downstream codon with first-position C (black) or U (gray). Shown are mean values from n = 3 independent biological replicates (circles) and mean values (solid line) with error bars representing SE. According to a two-tailed Welch’s t -test: *** P = 0.0002 ( t = 4.802; df = 16.71). Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: DNA was amplified by polymerase chain reaction (PCR) and transcribed by addition of T7 RNA polymerase (1.6 unit/μl), nucleotide triphosphates (ATP, NU-1010; GTP, NU-1012; CTP, NU1011; and either UTP, NU-1013, or m 1 ΨTP, NU-890; 3 mM each, Jena Bioscience), RNase inhibitor (0.2 units/μl, moloX GmbH), E. coli pyrophosphatase (0.005 units/μl, New England Biolabs, M0361) in buffer (40 mM Tris-HCl (Sigma-Aldrich, 252859) pH 7.5, 15 mM MgCl 2 (Sigma-Aldrich, M9272), 2 mM spermidine (Sigma-Aldrich, S2501), 10 mM NaCl (Sigma-Aldrich, S7653), 10 mM dithiothreitol (Roth, 6908-4)) and incubated at 37 °C for 3 h. The RNA product was purified by fast protein liquid chromatography in buffer (30 mM BisTris (Roth, 9140.3), pH 6, 1 mM EDTA (Merck, 137004)) using a linear gradient of 0.3–1.5 M NaCl, followed by ethanol precipitation.

Techniques: Sequencing, Fluorescence, Transfection, Modification, Variant Assay, Two Tailed Test