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. 2022 Feb 4;298(3):101657. doi: 10.1016/j.jbc.2022.101657

Figure 5.

Figure 5

In vitro investigation of Erg-Gly synthesis by ErgS.A, Erg-Gly synthesis reaction scheme (coupled reaction, when GlyRS and ErgS are present at the same time). Step 1: Gly activation into Gly∼AMP by GlyRS and step 2: transfer onto tRNAGly; step 3: Erg-Gly synthesis from Gly-tRNAGly. Colored bands below the scheme indicate up to which step the coupled reaction proceeds, when the indicated component (right side) is omitted (−) or added (+). Numbers refer to the lanes presenting the in vitro results in (C). B, left panel, [14C]Gly-tRNAGly synthesis by YliHis6-GlyRS or total aaRS extracts (containing GlyRS) from wt Sce in a typical coupled reaction. Results are identical for both sources of GlyRS (denoted by GlyRSa). Middle panel, control TLC showing mobility of synthetic Erg-Asp and Erg-Gly in the CHCl3:CH3OH:H2O (130:50:8, v:v:v) mobile phase. Right panel, control reactions with purified Afm ErdS (produces Erg-[14C]Asp, indicated with an asterisk, and a second band (∗∗) discussed below) and purified Yli MBP-ErgS with the indicated components. ErgS produces a compound (∗) with properties identical to synErg-Gly, showing that it synthesizes Erg-Gly. C, Erg-[14C]Gly synthesis experiments under the indicated conditions (+, presence; −, absence of the indicated components). Yli MBP-ErgS (or mutated ErgS) was added to [14C]Gly-tRNAGly produced by Sce GlyRS (in a total protein extract) or YliHis6-GlyRS in the presence/absence of various substrates and/or RNase A. Asterisks indicate position of Erg-[14C]Gly. D, Erg-[14C]Gly synthesis (step 3 only) with added [14C]Gly-tRNA (∼200 nM) and increasing amounts of cold Gly-tRNA (100–300 nM). E, isotopic competition of cold Gly for Erg-[14C]Gly synthesis. 15 [14C]aa-tRNAs were synthesized with 15 aaRSs (Sce total proteins), from 15 [14C]aa and total Sce tRNA, to which Erg, ErgS, the ErgSα(-)1 mutant, or RNase A was added. [14C]aa indicate bands attributed to free radiolabeled amino acids present in the reaction and separated by TLC. The red asterisk indicates Erg-[14C]Gly. In all cases, bars indicate standard deviation and the number of replicates is indicated.