tRNA purification

The E. coli tRNA_f^MetA₁–U₇₂ variant was overexpressed in PalΔmetZWVΔmetY::kan carrying pBSTNAV-tRNA_f^MetA₁–U₇₂ and purified as previously described (Mechulam et al. 2007). Briefly, a whole tRNA extract was prepared according to the Zubay procedure (Zubay 1962). The overexpressed tRNA_f^Met was then separated from other tRNAs by an anion exchange step on a Q-Hiload column (16 × 100 mm) equilibrated in 20 mM Tris–HCl, pH 7.6, 200 mM NaCl, 8 mM MgCl₂, 0.1 mM EDTA. tRNAs were eluted using a 350 mM to 550 mM NaCl gradient in the same buffer. About 10 mg of tRNA_f^MetA₁–U₇₂ was obtained from 1 L of E. coli culture. Methionine acceptance of the tRNA_f^MetA₁–U₇₂ preparation ranged between 1450 and 1600 pmol/A₂₆₀ unit. Before crystallization, the tRNA was methionylated and purified as described in Mechulam et al. (2007). The aminoacylated tRNA was resuspended in water (final concentration 400 µM) and diluted to 50 µM in a buffer containing 10 mM MOPS, pH 6.7, 5 mM MgCl₂, 200 mM NaCl, 10 mM 2-mercaptoethanol before crystallization trials.