In vitro translation of peuA mRNA.

(A) In vitro translation analysis of the +1 and +39 peuA transcripts labeled with the FLAG tag. The +1-peuA′-flag RNA and +39-peuA′-flag RNA were first synthesized by in vitro transcription, as described in the MATERIALS AND METHODS, and a mixture containing either the +1-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) or the +39-peuA′-flag RNA (30 pmol)/fur-flag RNA (3 pmol) as the template was subjected to in vitro translation. The FLAG-fused proteins translated were separated on 15% SDS-polyacrylamide gels, and were detected by western blotting using anti-FLAG IgG. (B) Confirmation of the presence of +1-peuA′-flag RNA and +39-peuA′-flag RNA in the reaction mixture for in vitro translation. These RNA fragments were detected in the reaction mixture by northern blotting using a DIG-labeled peuA probe.