Preparation of aminoacyl-tRNA for peptide formation assay

Yeast initiator tRNA_i^Met was prepared by T7 in vitro transcription. As noted previously, in vitro transcribed tRNA_i^Met lacking modified nucleotides readily functions in elongation (Astrom and Bystrom, Cell 1994. 79:535–546; Pestova and Hellen 2001. RNA 7:1496–1505; Pestova and Hellen 2003. Genes & Development 17:181–186). Yeast tRNA^Phe (Sigma) and tRNA^Lys (tRNAprobes, Texas) were obtained from commercial vendors.

The tRNA_i^Met was aminoacylated using purified His-tagged E. coli MetRS. E. coli XL1 Blue cells (Agilent) carrying the MetRS expression plasmid pRA101 (Alexander et al., 1998) were grown in 500 ml LB medium containing 100 µg/ml ampicillin at 37°C to A₆₀₀ = 0.5. Following addition of 0.2 mM IPTG the culture was incubated at 20°C for 14 hr. Following harvesting, the cell pellet was suspended in 20 ml lysis buffer (50 mM Tris-HCl [pH 7.5], 300 mM KCl, 6 mM 2-mercarptoethanol, and 10% glycerol) and cells were broken by sonication using a microtip (5 cycles of 30 s pulse followed by 30 s cooling at 4°C). The cell lysate was cleared by centrifugation at 27,000 x g for 30 min and then mixed gently with 1 ml Ni-NTA resin (Qiagen) at 4°C for 1 hr. The resin was transferred to a 1 ml disposable column (Qiagen), washed sequentially with 10 ml lysis buffer, 20 ml lysis buffer containing 20 mM imidazole, and then protein was eluted in 4 ml lysis buffer containing 250 mM imidazole. The elute was dialyzed overnight against 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 10 mM MgCl₂, 1 mM DTT and 10% glycerol. For aminoacylation of tRNA_i^Met, 5 µM tRNA_i^Met was mixed with 2 mM ATP, 0.3 µM [³⁵S]Met (Perkin Elmer), 10 mM MgCl₂ and 1 µM MetRS in reaction buffer (100 mM HEPES-KOH [pH 7.5], 10 mM KCl and 1 mM DTT), and then incubated at 37C for 30 min. Typically, about 60% of tRNA_i^Met was aminoacylated with [³⁵S]Met.

The tRNA^Phe was aminoacylated using purified yeast PheRS. To prepare PheRS, the open reading frames (ORFs) of FRS2 and FRS1, encoding the α and β subunits of yeast PheRS, respectively, were cloned into the polycistronic expression vector pST39 (Tan, 2001). First, an N-terminal His₆-tagged version of the FRS2 and FRS1 open reading frames were cloned between the NdeI and BamHI sites of the vector pET3a Trm, and then FRS2 was moved as an XbaI-BamHI fragment to the expression vector pST39 generating the plasmid pC5105. Next, the FRS1 open reading frame was transferred on a BspEI-MluI fragment to pC5105 generating the plasmid pC5106. For purification of PheRS, E. coli strain BL21(DE3) pLys (Agilent) carrying pC5106 was grown in 500 ml LB medium containing 100 µg/ml ampicillin at 37°C to A₆₀₀ = 0.5. Then, 0.2 mM IPTG was added and the culture was incubated at 25C for 16 hr. Following harvesting, the cell pellet was suspended in 20 ml lysis buffer (20 mM Tris-HCl [pH 7.5], 500 mM KCl, 5 mM MgCl₂, 1 mM 2-mercaptoethanol, 10 mM imidazole and 10% glycerol), and PheRS was purified as described above for purification of MetRS. The final PheRS elute was dialyzed overnight against 20 mM Tris-HCl (pH 7.5), 150 mM KOAc, 2.5 mM MgO(Ac)₂, 2 mM DTT and 10% glycerol.

To aminoacylate tRNA^Lys, we first cloned N-terminal His₆-tagged KRS1 (Lysyl-tRNA synthetase from S. cerevisiae) into pET3a between the NdeI and MluI restriction sites. For purification of LysRS, E. coli strain BL21(DE3) CodonPlus-RIL (Agilent) was transformed with pET3a-KRS1 and cells were grown in 250 ml LB medium containing 100 µg/ml ampicillin at 37°C to A₆₀₀ = 0.5. Following addition of 0.5 mM IPTG the culture was incubated at 20°C for 16 hr. LysRS was purified as described above for the purification of PheRS. Aminoacylation of tRNA_Phe and tRNA_Lys were performed as described previously (Gutierrez et al., 2013).