Figure 2.

In vitro reconstitution of Sgt2•TA^Cm complex. (A) Schematic depiction of the generation and purification of Sgt2•TA^Cm. In the presence of His₆-Sgt2, TA is synthesized and labeled with the fluorescent amino acid Cm using IVT in E. coli S30 extract coupled to amber suppression technology. Subsequent affinity purification generates the purified Sgt2•TA^Cm complex. (B) Sample SDS-PAGE analysis of amber suppression efficiency in a trial IVT reaction for a model TA substrate, Bos1. Reaction 1 contains a control plasmid that does not have the amber codon. Reaction 2 and 3 were carried out using the amber codon-containing plasmid in the absence or presence of Cm, CmRS and RF-1, respectively. This figure was adapted from Figure 5B in (Rao et al., 2016).