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. 2021 Jul 30;12:4644. doi: 10.1038/s41467-021-24911-1

Fig. 2. +1FS of E. coli tRNAPro(UGG) in a biochemical functional assay.

Fig. 2

a The reaction scheme of the functional assay to measure the yield of +1FS as the fractional conversion of fMP to fMPV. An E. coli 70SIC programmed with the non-slippery CCA-A reporter or the slippery CCC-A reporter was mixed with a TC containing EF-Tu-GTP with a native or transcript of tRNAPro(UGG), Val-tRNA (*UAC, *U = cmo5U anticodon), and Ser-tRNA (GCU anticodon) in the HF or CE buffer containing the indicated MgCl2 concentration. Each reaction was quenched after 5 min with 0.5 M KOH. Peptides were resolved by electrophoretic TLC and quantified by phosphor-imaging. b–f The fractional conversion of fMP to fMPV (pink) was reported for tRNAPro(UGG) in the native-state at the CCC-A codon motif at 37 °C (b); in the native-state at the CCC-A codon motif at 20 °C (c); in the native-state at the CCA-A codon motif at 20 °C (d); in the transcript-state at the CCC-A codon motif at 20 °C (e); and in the transcript-state at the CCA-A codon motif at 20 °C (f). All data are presented as mean ± SD. The bars in graphs b−f are SDs of three independent (n = 3) experiments; for datasets CCC-A transcript/HF(20),CE(3.5),CE(20); CCA-A transcript HF(3.5), HF(20),CE(3.5), n = 4.