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. 2019 Feb 11;15(2):e1007646. doi: 10.1371/journal.pgen.1007646

Fig 1. Characteristics of the speF locus.

Fig 1

(A) Schematic representation of the speF locus including orf34, speF and potE. The transcription start site (TSS) is indicated. The initiation codons of orf34 and speF are located at nucleotides 32 and 516 of the mRNA, respectively. The red vertical arrow indicates the site of rho-dependent premature transcription termination (T). Horizontal arrows indicate the short transcript obtained due to Rho (157 nt) and the full-length polycistronic mRNA. (B) Start site determination. The transcription start site of speF operon was determined by primer extension using primer 1614. RNA was extracted form cultures grown in LB to OD600 of 0.5. (C) The sequence of the transcript obtained by premature termination. The Shine-Dalgarno (AGGA), the initiation codon (AUG) and the stop codon (UGA) of orf34 are in magenta. The consecutive arginine codons AGG;CGG are in purple. UAA11 and UAA26 indicate mutated codons leading to premature translation termination and thus to premature transcription termination by Rho. (D) Translation of the entire orf34 prevents premature transcription termination by Rho. Cultures carrying Ptac-orf34-speF plasmids wild type and translational mutants as indicated (orf34AAA; orf34UAA11 and orf34UAA26), were grown in LB to OD600 of 0.2 and then treated with 20 μg/ml BCM for 60 minutes (+) or were left untreated (-). Northern blot of RNA samples (10 μg) separated using 6% urea-polyacrylamide gels. The membranes were probed with end-labeled orf34 (1614) and 5S rRNA (459) specific primers. 5S RNA serves as a loading control. The Full-length (FL) and the short transcript obtained by premature termination (T) are indicated in red.