Imagequant

Quantification of activity data was performed using ImageQuant (Cytiva Life Sciences, https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software), and data analysis (i.e., calculation of mean and standard error) was performed using PRISM (GraphPad Software, https://www.graphpad.com/scientific-software/prism/).

Cells from an overnight culture were diluted 300-fold into fresh LB (25 ml) containing Amp (100 μg/ml), Kan (50 μg/ml) and l-arabinose (5 mM) and grown for 4 h at 37°C. Cells were pelleted at 4°C, resuspended in Z buffer, transferred to a 1.5 ml Eppendorf tube, and then re-pelleted. TRIzol (Invitrogen) reagent (1 ml) was added, and the pellet was resuspended by pipetting and vortex mixing (5 min) at room temperature. Chloroform was added, mixed, then the aqueous phase was transferred to a new tube. RNA was precipitated with isopropanol then pelleted. The RNA pellet was washed with 70% ethanol and dissolved in water. Relative amounts of P-16S rRNA were determined using poisoned primer extension, essentially as previously described (44 (link),60 (link)). In a 20 μl reaction, primer #1456 (5′-[Cy5]-AAAGTGGTAAGCGCCCT-3′) (0.42 pmol) was incubated with RNA (5 μg) at 50°C for 10 min in AMV buffer (NEB), AMV reverse transcriptase (NEB, 3 units), ddATP, dCTP, dGTP, and dTTP (3.5 nmol each) were added, and the reaction was incubated at 42°C for 1 h. Products were resolved by denaturing 8% PAGE, gels were imaged using a Typhoon 5 (Cytiva), and data were quantified using ImageQuant (Cytiva).

70S ICs were prepared using E. coli ribosomes programmed with various mRNAs and f-[³⁵S]-Met-tRNA^Met in the P site (70 (link)). Translation was initiated by mixing equal volumes of TC (20–60 μM aatRNA(s), 24 μM EF-G, 60 μM EF-Tu) with ICs (140 nM) in 219-Tris buffer (50 mM Tris pH 7.5, 70 mM NH₄Cl, 30 mM KCl, 7 mM MgCl₂, and 5 mM βME). All initiation factors (IF-1, IF-2, and IF-3) and translation factors (EF-Tu and EF-G) used were His-tag purified from E. coli using plasmids available from AddGene (26 (link)). The reactions were quenched with equal volume of 1 M KOH at discrete time points (0 s–20 min) by hand (5 s–20 min) or using a KinTek RQF-3 quench flow apparatus (0.001–5 s) (71 (link)). Each sample was diluted 1:10 in nuclease-free water, and the reactants, intermediates, and products were separated by eTLC, visualized by phosphorimaging, and quantified with ImageQuant (Cytiva Life Sciences) as previously described (26 (link)). Depending on the expected peptide products, eTLCs were run in different running buffer conditions to improve separation (26 (link)). eTLCs analyzing peptides containing one or more lysines were run in pyridine acetate buffer, pH 2.8, while eTLCs separating peptides with valine (but no lysine) were run in pyridine acetate buffer, pH 5.2. For m⁶A studies in this work, all m⁶A mRNA constructs were purchased from Dharmacon, Horizon Discovery.

Five microliters of purified FakB1 (40 μM) was added to 20 μl of high-capacity streptavidin agarose beads (Thermo Fisher) and 25 μl of liposomes containing the following: 1 mol percent of DOPE-N-(cap biotinyl) and 99 mol percent of DOPC or DOPG; 1 mol percent of DOPE-N-(cap biotinyl), 89 mol percent DOPG, and 10 mol percent of [¹⁴C]16:0; 1 mol percent of DOPE-N-(cap biotinyl), 79 mol percent DOPG, and 20 mol percent of [¹⁴C]16:0. Buffer (0.1 M Tris-HCl, pH 7.5) was added to a total volume of 200 μl, and samples were incubated at room temperature on a shaker for 1 h before being centrifuged at 2000g for 5 min. Supernatant was decanted without disturbing the streptavidin beads, and excess moisture was wicked from the beads before resuspending in an equal volume of buffer. A Western blot was performed on aliquots from the supernatant and pellet using monoclonal anti-polyHistidine−alkaline phosphatase antibody (Sigma). Blots were images on a Typhoon FLA 9500, and band intensities were quantified using ImageQuant (Cytiva). Statistical significance was determined using the two-tailed Student’s t test.

HEK 293T (RRID: CVCL_0063) cells (ATCC, USA) were maintained in RPMI-1640 (#SH30027.01, Cytiva, USA) and supplemented with 10% fetal bovine serum (#10437028, Gibco, ThermoFisher, USA) at at 37 °C with 5% CO₂ in a humidified atmosphere. The deaminase activity was performed as previously described⁵⁵ . Lysate was placed on ice and immediately used for the deaminase assay. Inhibitor oligos (and controls) were heated to 80°C for 5 minutes, and then cooled to RT to induce hairpin formation. They were then incubated at varying concentrations in 5 μL with 10 μL of cell lysate at 37°C for 15 minutes to promote binding of oligos to A3A. To this, 5 μL of: 0.25 μL RNAse A, 800 nM fluorescent oligo, 10x UDG buffer, and 0.25 μL UDG were added to each sample for a total volume of 20 μL and incubated at 37°C for 1 hour. The fluorescently-labeled oligo has the following sequence: (5′-ATTATTATTATTCGAATGGATTTATTTATTTATTTATTTATTT-fluorescein-3′). The samples were run on a 15% Urea-TBE acrylamide gel, imaged with a Typhoon FLA-7000 imager (GE Healthcare), and then quantified using ImageQuant (Cytiva, USA).

HEK 293 T (RRID: CVCL_0063) cells (ATCC, USA) were maintained in RPMI-1640 (#SH30027.01, Cytiva, USA) supplemented with 10% fetal bovine serum (#10437028, Gibco, ThermoFisher, USA) at 37 °C with 5% CO₂ in a humidified atmosphere. The ssDNA deaminase activity was performed as described in ref. ^{71 (link)}. Whole cell lysates were prepared, placed on ice, and immediately used for the deaminase assay. Inhibitor oligos (and controls) were heated to 80 °C for 5 min, and then cooled to RT to induce hairpin formation. They were then prepared at varying concentrations in 5 µL and combined with 10 µL of cell lysate at 37 °C for 15 min to promote binding of oligos to A3A. To this reaction, 5 µL of a mastermix containing 0.25 µL RNAse A, 800 nM fluorescent ssDNA substrate, 10x UDG buffer (NEB #M0280), and 0.25 µL UDG (NEB #M0280) were added to each sample for a total volume of 20 µL and incubated at 37 °C for 1 h. The fluorescently-labeled oligo has the following sequence: (5′-(ATT)₃ATTCGAATGG(ATTT)₆-fluorescein-3′). Reactions were fractionated on a 15% Urea-TBE acrylamide gel, imaged with a Typhoon FLA-7000 imager (GE Healthcare), and then quantified using ImageQuant (Cytiva, USA).

ECs were assembled and immobilized as described (38 (link)). Sequences of the oligonucleotides used for the assembly of ECs are shown on Fig.4C. For assembly of ECs used for experiments on Fig. 4C, 13 nt long RNA was radiolabelled at the 5’-end with [γ-³²P] ATP and T4 Polynucleotide kinase (New England Biolabs) prior to complexes assembly. Stalled elongation complexes EC14, EC15 and EC16 were obtained by extension of the initial RNA13 in EC13 with 10 μM NTP sets according to the sequence for 5 min and then were washed with TB to remove Mg²⁺ and NTPs. Reactions were initiated by addition of 10 mM MgCl₂ with or without either 1 μM NTPs or 250 μM PPi. Single nucleotide addition and pyrophosphorolysis experiments were performed at 30°C in transcription buffer (TB) containing 20 mM Tris–HCl pH 6.8, 40 mM KCl, 10 mM MgCl₂, transcript hydrolysis was done in the same buffer except at pH 7.9. After incubation for intervals of time specified on Figures, reactions were stopped with formamide-containing buffer. Products were resolved by denaturing 23% polyacrylamide gel electrophoresis (PAGE) (8 M Urea), revealed by PhosphorImaging (Cytiva) and visualized using ImageQuant (Cytiva) software. Kinetics data were fitted to a single exponential equation y=y₀+a^−bx using SigmaPlot software by non-linear regression to determine rate constants of the reactions.