Typhoon trio phosphorimaging system

Gel electrophoresis experiments were performed with 20% native gels (19:1 acrylamide:bis-acrylamide). To ensure G4DNA maintained intact during the run, the gel and 1× TBE running buffer (89 mM Tris Borate pH 8.3 and 2 mM Na₂ EDTA) were supplemented with 20 mM KCl. The 2 nM radiolabeled oligonucleotides were prepared in 10 mM Tris, 1 mM EDTA, and with/without 140 mM KCl and 8 mM MgCl₂. Oligonucleotides in 10 mM Tris and 1 mM EDTA were heated and flash cooled. Oligonucleotides in 10 mM Tris, 1 mM EDTA, and with 140 mM KCl and 8 mM MgCl₂ were heated at 95°C and slowly cooled to room temperature. Samples were resolved by electrophoresis for 8 hours, visualized using a Typhoon Trio phosphorimaging system, and purity quantified using ImageQuant software (GE Healthcare).

30S substrate methylation was determined using a reverse transcription (RT) assay with E. coli MRE600 30S subunits purified as described previously (30 (link)). Various concentrations of methyltransferase protein (10–1000 pM) were incubated with a fixed amount of 30S (100 pM) for 1 hour at 37°C in 30S methylation assay buffer (10 mM HEPES–KOH (pH 7.5), 10 mM MgCl₂, 50 mM NH₄Cl, and 5 mM β-mercaptoethanol) containing SAM (1 mM). The reaction was terminated by phenol/chloroform extraction followed by ethanol precipitation to recover 16S rRNA. The reaction product was analyzed by RT using a ³²P-labeled DNA primer complementary to E. coli 16S rRNA nucleotides 1457–1473. Extension products were run on 10% PAGE-urea gels and visualized using a Typhoon Trio phosphorimaging system (GE Healthcare). Methylation of A1408 at the N1 position (m¹A1408) produces a strong stop in the RT reaction resulting in an intense band on the gel at the position corresponding to nucleotide C1409. Band intensities at different protein concentrations were determined using ImageQuant TL Software (GE Healthcare) and these values converted to fraction methylated, assuming complete methylation occurs in the reaction with 10-fold excess enzyme.