Translation of model peptides

Translation was carried out in a cell-free coupled transcription–translation FIT system²³. The reactions contained only five ARSs (MetRS, LysRS, GlyRS, AspRS and TyrRS) and five amino acids (Met, Lys, Gly, Asp and Tyr) together with E. coli total tRNA; under these conditions, free Pro is not charged onto the natural tRNA^Pro isoacceptors by ProRS. Transcription of the DNA templates into mRNAs was carried out by T7 RNA-polymerase present in the FIT system. Reactions were carried out at 37 °C for 20 min, except for the time-course analysis in 2.5 μl reaction mixture consisting of the following reagents: 50 mM HEPES-KOH (pH 7.6), 100 mM potassium acetate, 12.3 mM magnesium acetate, 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 20 mM creatine phosphate, 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolic acid, 2 mM spermidine, 1 mM DTT, 1.5 mg ml⁻¹ E. coli total tRNA, 1.2 μM E. coli ribosomes, 0.6 μM methionyl-tRNA formyltransferase, 2.7 μM IF1, 0.4 μM IF2, 1.5 μM IF3, 0.26 μM EF-G, 10 μM EF-Tu, 0.66 μM EF-Ts, 0.25 μM RF2, 0.17 μM RF3, 0.5 μM RRF, 4 μg ml⁻¹ creatine kinase, 3 μg ml⁻¹ myokinase, 0.1 μM inorganic pyrophosphatase, 0.1 μM nucleotide diphosphate kinase, 0.1 μM T7 RNA polymerase, 0.13 μM AspRS, 0.09 μM GlyRS, 0.11 μM LysRS, 0.03 μM MetRS, 0.02 μM TyrRS, 0.05 mM [¹⁴C]-aspartic acid, 0.5 mM glycine, 0.5 mM lysine, 0.5 mM methionine, 0.5 mM tyrosine and 50 μM aminoacyl-tRNA that is under investigation, where indicated, 3 μM lysylated EF-P and 0.5 μM DNA template (5′- GGCGT AATAC GACTC ACTAT AGGGT TAACT TTAAG AAGGA GAAAA ACATG AAGAA GAAGX XXXXX GGTGA CTACA AGGAC GACGA CGACA AGTAA GCTTC G -3′). For testing native tRNA^Pro, 50 μM native or transcribed [¹⁴C]Pro-tRNA^{Pro1/Pro2/Pro3} mixture and 0.5 mM cold aspartic acid were added instead of cold Pro-tRNA and [¹⁴C]-aspartic acid. The reactions were stopped by addition of an equal volume of stop solution (0.9 M Tris-HCl (pH 8.45), 8% SDS, 30% glycerol and 0.001% xylene cyanol) and incubation at 95 °C for 2 min, and then analysed by 15% tricine SDS–PAGE and autoradiography. Intensity bands of interest were normalized to the total [¹⁴C]-aspartic acid intensity (125 pmol) included in the reaction mixture.

For translation of FLK-FLAG, 0.5 μM template DNA coding for FLK-FLAG (5′- GGCGT AATAC GACTC ACTAT AGGGT TAACT TTAAG AAGGA GAAAA ACATG ATACA ACCTA TTTCC GGCCC TCCTC CTGGG CAACC ACCAG GTCAG GGAGA TAATC TGGAC TACAA GGACG ACGAC GACAA GTAAG CTTCG -3′), 0.38 μM AsnRS, 0.06 μM GlnRS, 0.4 μM IleRS, 0.04 μM LeuRS, 0.04 μM SerRS and 0.5 mM each of asparagine, glutamine, isoleucine, leucine and serine were added to the above reaction mixture. Wild-type or C13G Pro-tRNA^{Pro1/Pro2/Pro3} mixture was used for Pro incorporation.