Fig. 3: Activity and light response of NmPAL variants.

a By embedding a specific aptamer (denoted 04.17) near the Shine-Dalgarno sequence (SD) of an mRNA encoding DsRed, the expression of the fluorescent reporter can be modulated with NmPAL as a function of blue light⁴⁶. In darkness, NmPAL shows little affinity for the aptamer, and expression ensues. Under blue light, NmPAL binds and thus attenuates expression. b E. coli cultures harboring different NmPAL variants and the reporter system depicted in panel a were cultivated in darkness (black dots and gray bars) or under blue light (white dots and blue bars). Normalized DsRed fluorescence represents mean ± s.d. of at least three biologically independent samples. c NmPAL variants were expressed in HeLa cells to translationally repress expression of a luciferase reporter, conceptually similar to the setup shown in panel a but using the 53.19 aptamer⁴⁶. Bars represent the mean ± s.d. of luminescence acquired for six biologically independent samples incubated in darkness (black dots and gray bars) or under blue light (white dots and blue bars). UTR5 refers to the intact reporter system giving rise to NmPAL-mediated light responses;⁴⁶ in M21, NmPAL binding is disrupted by a mutation in the target aptamer, and light responsiveness is abolished. As positive and negative controls, luciferase was constitutively expressed (Luc) or left out altogether (-Luc). d Ratio of the luminescence values obtained under light and dark conditions. e The interaction of wild-type NmPAL with the TAMRA-labeled 04.17 aptamer was assessed in its dark-adapted (black dots) and light-adapted states (white dots) by fluorescence anisotropy⁴⁶. The line represents a fit to a single-site binding isotherm. f As in e but for NmPAL Q347L. Experiments in panels e and f were repeated twice with similar results.