Figure 2
From: Enabling stop codon read-through translation in bacteria as a probe for amyloid aggregation
Design of the WH1(R0-4)-RF1 chimeras and the reporter lacZ-WT/amber plasmids. (a) The plasmid-borne fusions of the RepA-WH1 amyloidogenic peptides to E. coli RF1 (prfA gene). The control WH1(R0)-RF1 fusion (top) encodes at the N-terminus a His6-tag (green), two unique restriction sites and a flexible long linker (black) just before RF1 (red). The WH1(Rn)-RF1 chimeras accommodate the hydrophobic RepA-WH1 peptide repeats (blue; encircled: the hyper-amyloidogenic A31V mutation)22. Triplets encoding the Gly residues required to build the turn between the amyloidogenic repeats are also highlighted (orange). Just the single peptide repeat (R1, middle) and the final four repeats (R4, bottom) are displayed. (b) Arabinose-promoted expression (pBAD) cassette (left) with a wild-type lacZ reporter (blue) (top) or a mutated lacZ-amber variant carrying a premature UAG stop codon (bottom). (c) Functional assays in an E. coli MRA8-∆lacZ, prfA ts background. Complementation by the lacZ-WT construct leads to blue coloured bacteria (left). On the contrary, the expression of the lacZ-amber variant was unable to complement, due to the premature end of lacZ-amber translation (right). Experiment carried out at 30 °C because, in the absence of the WH1(Rn)-RF1 chimeras, translation termination is dependent on the host RF1ts.
