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. Author manuscript; available in PMC: 2013 Dec 1.
Published in final edited form as: Mol Microbiol. 2012 Oct 12;86(5):1167–1182. doi: 10.1111/mmi.12050

Figure 5. Effect of SD2 disruption on the 5' end of esp mRNA.

Figure 5

(A) Ribonuclease protection analysis (RPA) of processed esp mRNAs. Upper panel: Region complementary to the 382-nucleotide probe used for RPA. The arrow marks the approximate site of endonucleolytic processing by RNase E. Lower left panel: RPA of RNA from EHECΔLEE cells containing plasmid pPL1 (wild-type SD2 element) or pmut-SD2 (mutated SD2 element) (see Fig 3A). Total RNA from yeast was used as a negative control. Asterisks mark some of the processed transcripts. A triangle marks a processed species that is almost absent when SD2 is disrupted. A dot marks the unprocessed precursor. Lower right panel: E. coli K-12 carrying either a wild-type (wt) or temperature-sensitive (ts) RNase E allele was transformed with plasmid pmut-SD2. RNA samples were collected at a non-restrictive temperature (time 0) and at different time intervals after shifting the culture to the restrictive temperature and subjected to RPA.

(B) Location of the 5' ends of processed esp mRNAs in EHECΔLEE(pPL1-SD2). The products of 5' RLM-RACE were cloned and sequenced. *** indicates a cluster of sites where most of the 5' ends were located (18 out of 26 clones, highlighted in gray), corresponding to RPA products 130–140 bases long. ** indicates 5' ends (1 out of 26 clones for each underlined nucleotide) that correspond to RPA products 179–186 bases long. * indicates 5' ends (1 out of 26 clones for each underlined nucleotide) that correspond to RPA products 206–210 bases long. The 5' end represented by another clone corresponds to an upstream thymidine (triangle) and approximately matches the size of the RPA product labeled with a triangle in A. Two other clones have 5' ends located in the espA coding region downstream of the sequence shown in the figure. The SD2 and SDA sequences and start codons are shown in capital letters. The stop codons for sepL (TGA) and for the mini-ORF (TAA) are marked with a line above them. The arrows delimit the maximum region complementary to the RPA probe.