Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis

Journal: Nucleic Acids Research

Article Title: An RNA structural switch controlling bacterial toxin translation

doi: 10.1093/nar/gkag240

Figure Lengend Snippet: FASTBAC-Seq identifies single point loss-of-function (LOF) mutations in timP mRNA. ( A ) Schematic representation of the timP 5′UTR secondary structure in its active and inactive conformation. Yellow: the pseudoknot-forming sequences; gray: the TimR-binding site; blue: SD and start codon. ( B ) Schematic representation of the FASTBAC-Seq method. The timP gene from Salmonella enterica , flanked by either a constitutive promoter or a promoter-less sequence and homologous recombination sites (brown boxes), was amplified under error-prone conditions and recombined into the Escherichia coli chromosome. DNA was extracted from the resulting colonies, followed by polymerase chain reaction (PCR) amplification, high-throughput sequencing and statistical analysis to identify LOF mutations (see the “Materials and methods” section for details). ( C ) LOF point mutations identified by FASTBAC-Seq throughout the timP mRNA sequence. ( D ) LOF mutations identified at the start and stop codons of timP . ( E ) LOF mutations identified in the timP pseudoknot. N/A: not applicable. Green color indicates LOF mutations.

Article Snippet: A DNA sequence encoding timP mRNA from S. enterica serovar Typhimurium, preceded either by the PJ23106 promoter [ ], or by an incomplete version of the native timP promoter encompassing only the −10 box, and flanked with homology regions for homologous recombination in E. coli , were synthesized by Eurofins (GLR11 and GLR12, respectively; ).

Techniques: Binding Assay, Sequencing, Homologous Recombination, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing

Journal: Nucleic Acids Research

Article Title: An RNA structural switch controlling bacterial toxin translation

doi: 10.1093/nar/gkag240

Figure Lengend Snippet: LOF mutations in the timP 5′UTR abolish translation. The two top panels show Western blot (WB) analysis of TimP 3xFLAG expression in the indicated LOF mutants. The timP variants were expressed from an arabinose-inducible promoter on a plasmid. GroEL served as a loading control. The two bottom panels show Northern blot (NB) analysis of timP 3xflag mRNA levels for the indicated LOF mutants. Probing of 5S rRNA served as loading control.

Article Snippet: A DNA sequence encoding timP mRNA from S. enterica serovar Typhimurium, preceded either by the PJ23106 promoter [ ], or by an incomplete version of the native timP promoter encompassing only the −10 box, and flanked with homology regions for homologous recombination in E. coli , were synthesized by Eurofins (GLR11 and GLR12, respectively; ).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Northern Blot

Journal: Nucleic Acids Research

Article Title: An RNA structural switch controlling bacterial toxin translation

doi: 10.1093/nar/gkag240

Figure Lengend Snippet: An interaction between a previously predicted single-stranded region and SL4 is required for timP translation. ( A ) Secondary structure representation of the timP 5′UTR. Mutations designed to disrupt and restore the respective interactions are highlighted in red. ( B ) Western (WB) and Northern blots (NB) monitoring expression from the indicated timP 3xflag constructs. The timP variants were expressed as in Fig. . An unspecific band served as loading control for Western blot. Probing of 5S rRNA served as loading control in the Northern blot. ( C ) Toxicity resulting from arabinose-induced overexpression of wild-type or mutant timP . ( D ) Secondary structure representation of timP SL4. Mutations predicted to destabilize SL4 are highlighted in red. ( E ) Western blot monitoring TimP 3xFLAG expression. The tested timP variants were expressed from an arabinose-inducible promoter on the chromosome. GroEL served as loading control. ( F ) Quantification of heteroduplex formation between an antisense oligo and the SD sequence as a function of RNase H cleavage. Cleavage of the indicated mutants was normalized relative to wild type. Statistical analysis was performed using two-tailed t -test, *** P < .0005, ** P < .005, * P < .05, ns: nonsignificant.

Article Snippet: A DNA sequence encoding timP mRNA from S. enterica serovar Typhimurium, preceded either by the PJ23106 promoter [ ], or by an incomplete version of the native timP promoter encompassing only the −10 box, and flanked with homology regions for homologous recombination in E. coli , were synthesized by Eurofins (GLR11 and GLR12, respectively; ).

Techniques: Western Blot, Northern Blot, Expressing, Construct, Control, Over Expression, Mutagenesis, Sequencing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: An RNA structural switch controlling bacterial toxin translation

doi: 10.1093/nar/gkag240

Figure Lengend Snippet: Site 1–2 interaction determines the inactive conformation of timP 5′UTR. ( A ) Secondary structure representation of the timP 5′UTR. Mutations designed to disrupt and restore the respective interactions are highlighted in red. ( B ) Alignment of complementary regions of sites 1, 2, 3, and 4 in the indicated enterobacterial species, S. enterica serovar Typhimurium, Serratia proteamaculans, R. aquatilis . Nucleotides in red highlight changes compared to the Salmonella sequence. ( C ) RNase T1 cleavage of in vitro transcribed timP and indicated mutants in the presence or absence of TimR. ( D ) Quantification of RNase T1 cleavage at position G76 based on two independent experiments. For each lane, the band intensity at position G76 was first normalized to that of position G131. Then, for each timP variant, values were divided by the timP sample lacking TimR (control). Average values and standard deviation are shown. ( E ) Western blot monitoring TimP 3xFLAG expression. The timP variants were expressed from an arabinose-inducible promoter on a plasmid. GroEL served as loading control.

Article Snippet: A DNA sequence encoding timP mRNA from S. enterica serovar Typhimurium, preceded either by the PJ23106 promoter [ ], or by an incomplete version of the native timP promoter encompassing only the −10 box, and flanked with homology regions for homologous recombination in E. coli , were synthesized by Eurofins (GLR11 and GLR12, respectively; ).

Techniques: Sequencing, In Vitro, Variant Assay, Control, Standard Deviation, Western Blot, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: An RNA structural switch controlling bacterial toxin translation

doi: 10.1093/nar/gkag240

Figure Lengend Snippet: Schematic model for posttranscriptional regulation of the timPR T1TA system. The translationally inactive conformation of the timP 5′UTR is characterized by the interaction between sites 1 and 2, and SL4, which prevents 30S access to the SD. A structural rearrangement results in the formation of a pseudoknot structure between sites 1 and 3, which in turn allows site 2 to bind to site 4 to release the SD sequence from SL4. This permits 30S binding to the RBS and synthesis of the TimP toxin. Alternatively, the sRNA TimR binds on SL2, which abolishes pseudoknot formation and results in inhibited translation.

Article Snippet: A DNA sequence encoding timP mRNA from S. enterica serovar Typhimurium, preceded either by the PJ23106 promoter [ ], or by an incomplete version of the native timP promoter encompassing only the −10 box, and flanked with homology regions for homologous recombination in E. coli , were synthesized by Eurofins (GLR11 and GLR12, respectively; ).

Techniques: Sequencing, Binding Assay