FIGURE 3.

Analysis of ribonuclease activity by YafQ in vitro. A and B, YafQ, YafQ mutants (H87Q, H23A, and K48A), DinJ-YafQ, and DinJ-YafQ mutants (F49A and I59N/I62N) cleaved total RNA in vitro. Each reaction (10 μl) contained 5 μg of total RNA and 0.5 μg of protein in the reaction buffer 10 mm Tris-Cl (pH 8.0) and was incubated for 15 min at 37 °C. Samples were fractionated on a 6% polyacrylamide gel. C, superimposition of the free YafQ structure and the YafQ in complex. Loop α3-β2 in free YafQ undergoes a major conformational change upon interaction with DinJ. D, EMSAs with decreasing concentrations of DinJ-YafQ, DinJ(F49A)-YafQ, or DinJ(I59N/I62N)-YafQ were performed with pal I-II (0.5 μm). Samples were fractionated on a 1% agarose gel. Triangle, upshifted band. All gels were visualized by ethidium bromide staining.