Figure 5.

Dark-induced activation of endonucleolytic cleavages upstream of the stem–loop structure. In vitro processing analysis with the dark and the light extracts in the presence of 5 µg of tRNA. Five micrograms of the light- and dark-processing extract proteins were incubated with a uniformly labeled or 5′-labeled petD 3′-UTR probe in the presence of 5 µg of tRNA. After the given times (bottom) the reaction was stopped and RNA products analyzed by denaturing TBE gel electrophoresis and subsequent autoradiography. The 5′-labeled probe was incubated with 5 µg of the light-extract proteins in the absence of tRNA to control the processing activity of this extract on 5′-monophosphate-carrying RNAs. To check for the endonucleolytic cleavage sites used by the dark extract the 5′-labeled probe was incubated with 5 µg of the dark extract in the presence of tRNA. RNA products were analyzed as described above.