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. 2019 Jun 7;15(6):e1007813. doi: 10.1371/journal.ppat.1007813

Fig 2. Influence of CsrA on lcrF transcript translation.

Fig 2

(A) Upper panel: Overview of the yscW-lcrF locus and the generated lcrF transcripts used for the RNA binding assay below and in (B); red bars: CsrA binding sites. The yscW-lcrF promoter is indicated by a broken arrow; the identified CsrA binding sites are indicated with red bars. The numbers give the nucleotide positions with respect to the translation initiation site of lcrF. Lower panel: RNA binding assays with increasing concentration of purified CsrA-His6 to lcrF transcripts harboring the region of position -123 to +75 (left panel) or -123 to +15 (right panel) with respect to the translational start site. The CsrA-lcrF mRNA complex is indicated by a star. c: hns control transcript. (B) The lcrF transcript (-123 to +75) and a mutant transcript in which the GGA motif is exchanged against TTC were incubated with increasing concentration of purified CsrA-His6. The CsrA-lcrF mRNA complex is indicated by a star. (C) To study CsrA influence on translation initiation of lcrF, expression of an arabinose inducible translational lcrF-lacZ fusion encoded on pKB14 and a mutant variant with an open lcrF RNA thermometer element encoded on pKB99 was analyzed in YPIII (WT) pV (empty cloning vector pHSG576) and YP53 (ΔcsrA) pV and YP53 pKB60 (csrA+). The bacteria harboring the different lcrF-lacZ fusions were grown at 25°C to exponential phase and were then shifted for 4 h to 37°C in the presence of 0.1% arabinose and absence of Ca2+. β-galactosidase activities represent the mean ± SD from three independent biological replicates which were analyzed with Student’s t-test. *: P<0,05: **: P<0,01, ***: P<0,001. (D) An amplified lcrF DNA fragment of pMP1 was used for in vitro transcription. The resulting lcrF mRNA fragment was used for in vitro translation in the absence or presence of CsrA. Samples were analyzed by western blotting with a polyclonal antibody directed against LcrF. An assay without the lcrF template RNA was used as negative control. c: loading control (lower panel).