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. 2009 Feb 12;4(2):e4459. doi: 10.1371/journal.pone.0004459

Figure 1. Construction of ssrA-DD mutant of S. coelicolor ssrA gene and detection of tagged proteins.

Figure 1

A. Detail of the sequence and proposed secondary structure of the tag-coding sequence of S. coelicolor tmRNA. Bases in red in ssrA-DD were altered by mutagenesis. B. Northern blot analysis of total RNA isolated from empty vector transformed cells (C = control), and cells expressing tmRNA from a single-copy (S) and multi-copy (M) plasmid carrying ssrA-DD. Ethidium bromide stained ribosomal RNA (rRNA) is shown as a loading control. Note that the probe is generated from full-length ssrA DNA and does not distinguish between the wild type and mutant tmRNA. C. Western blot detection of tmRNADD tagging in S. coelicolor. Total proteins from strains analyzed in panel B were separated by SDS-PAGE and either stained with Coomassie blue (CB) or transferred to PVDF membranes and probed with affinity-purified anti-ssrA-DD tag antibody (WB). Immunocomplexes were detected by ECL. D. Tagging in S. lividans analyzed as in panel C.