Figure 4.

ThT is available for measuring RNA metabolic enzyme activities in vivo. (A) Experimental procedures are schematically described. Overnight culture of Escherichia coli strains were diluted 100-fold into fresh LB medium containing kanamycin (Km) and incubated until OD₆₆₀ reached to 0.4–0.5. Subsequently the culture was supplemented with 1 mM IPTG and the expression of PNPase variants were induced at 37°C for 5 h. The culture was divided into two tubes; one of them was incubated on ice for 19 h and the other was supplemented with 200 μg/ml rifampicin (Rfp) and incubated at 37°C for 19 h. After removal of culture supernatant, equal wet weights of cells were suspended into PBS containing 25 μM ThT. ThT fluorescent spectra of the suspensions were measured using a spectrofluorometer (B), and cells were observed under fluorescence microscopy (C). (B) Data points represent the means and standard deviations of results from three independent experiments. The standard deviation is less than that corresponding to the size of the symbol if no error bars are seen. (C) Scale bars indicate 10 μm. (D) Expression of PNPase variants were checked by SDS-PAGE with CBB staining. Positions of molecular mass markers are represented at the left of the panel.