Identification of RNase III cleavage sites in <i>betT</i> mRNA <i>in vitro</i> and <i>in vivo</i>.

<p>(A) <i>In vitro</i> cleavage of the full-length synthetic <i>betT</i> mRNA. The 5′-end-labeled <i>betT</i> transcript (4 pmol) was incubated with purified RNase III (1 pmol) in cleavage buffer with (+) or without (-) MgCl₂ at 37°C. The size of the cleavage products was estimated using size markers generated by internally labeled transcripts. The major cleavage products are indicated with arrows. Other minor cleavage products are indicated with asterisks. (B) Primer extension analysis of <i>betT</i> mRNA. Total RNA was prepared from MG1655<i>rnc</i> harboring pBetRS1 and either pKAN6B or pRNC1, which exogenously overexpressed <i>betT</i> mRNA, hybridized with a 5′-³²P-end-labeled primer (betT+120R), and extended using AMV reverse transcriptase. Sequencing ladders were produced using the same primer used in cDNA synthesis and PCR DNA, encompassing the <i>betT</i> gene as a template. (C) The predicted secondary structure of <i>betT</i> mRNA region encompassing RNase III cleavage sites. The secondary structure was determined using the M-fold program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100520#pone.0100520-Zuker1" target="_blank">[33]</a>. (D) <i>In vitro</i> cleavage of the model hairpin RNA of <i>betT</i> mRNA. 3′-end-labeled <i>betT</i> model hairpin (25 pmol) was incubated with purified RNase III (1 pmol) in a cleavage buffer with (+) or without (−) MgCl₂, respectively. Cleavage products (I, II, III, and IV) were identified using size markers generated by alkaline hydrolysis and RNase T1 digestion. Relative abundance of each cleavage product was assessed by measuring the radioactivity of each band and plotted.