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. 2016 Dec 12;45(7):4081–4093. doi: 10.1093/nar/gkw1229

Figure 3.

Figure 3.

Activity of V-Spinach tRNA in protein synthesis. (A) Suppression of the lacZ125am locus in E. coli CA244 cells (Yale CGSC stock center) by V-Spinach tRNATyr with the 5΄-CUA anticodon (CUA), but not by the Spinach stand-alone, V-Spinach tRNATyr with the 5΄-GUA anticodon (GUA), or T- or D-Spinach tRNATyr with the CUA anticodon (CUA). E. coli cells expressing each Spinach construct were streaked out on an LB plate with ampicillin. Three to five single colonies with each construct were picked and re-streaked to 1 cm length on a minimal M9 plate with ampicillin and X-gal. This single-colony isolation ensured the homogeneity of each clone of cells. Representative streaks on M9 plates are shown. (B) Activity of the wild-type (WT) Ec tRNATyr and V-Spinach Ec tRNATyr in major steps of protein synthesis, including aminoacylation, stability of the ternary complex, accommodation to the ribosome A-site, and formation of the first peptide bond on an mRNA template with the sequence starting with 5΄-AUG-UAU-3΄. Both V-Spinach tRNATyr and Ec tRNATyr, each containing the natural Tyr anticodon 5΄-GUA, were isolated from E. coli cells and purified to homogeneity. (C) Suppression of the amber codon 5΄-UAG at the lacZU118Am locus by the amber suppressor form of V-Spinach tRNATyr harboring the anticodon 5΄-CUA, leading to synthesis of β-gal. Measurement of β-gal activity encoded in XAC-1 cells expressing a plasmid-borne amber suppressor form of tRNATyr (Tyr), amber suppressor form of V-Spinach tRNATyr (Spinach), or none. Each activity is reported as a fraction relative to the activity encoded by the lacZWT gene (no amber mutation) in XAC-1 cells. Error bars denote SD (standard deviation), n = 3. (D) Suppression of trmDAm (Y19) by the amber suppressor form of V-Spinach tRNATyr leads to synthesis of the growth-dependent TrmD enzyme, which supports E. coli cell viability. The trmDAm (Y19) locus contains an amber codon at the Y19 position, which prevents translation of the trmD gene. This study was conducted in E. coli trmD-knockout (trmD-KO) cells where the chromosome-encoded trmD gene was disrupted with an antibiotic marker and cells were maintained viable in the presence of arabinose by a plasmid-borne and arabinose-controlled expression of the human counterpart trm5 gene (42). (E) Examination of viability of E. coli trmD-KO cells expressing a plasmid-borne trmDWT (row 1), a plasmid-borne trmDAm (Y19) (row 2), a plasmid-borne trmDAm (Y19) and the amber suppressor gene of Ec tRNATyr (row 3), and a plasmid-borne trmDAm (Y19) and the amber suppressor gene of V-Spinach tRNATyr (row 4) in the presence or absence of arabinose (Ara).