Journal: Scientific Reports
Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli
Figure Lengend Snippet: Restoration of RNase III-dependent regulation of eno - cat expression by putative cis -antisense RNA expression. ( a ) Schematic diagram showing the transcriptional initiation and termination sites (TISs and TTSs, respectively) of putative cis -antisense RNA. The 5′ and 3′ termini of putative cis- antisense RNAs that were inferred from cDNAs were located at positions +451, +272, −36, and −83 (designated as TIS1, TIS2, TIS3, and TIS4, respectively) from the start codon of the eno coding region, and at positions +1,419, +1,265, +736, and +696 from the start codon of the pyrG coding region (designated as TTS1, TTS2, TTS3, and TTS4, respectively). The secondary structure of the TTSs was inferred using the M-fold program. ( b ) Effects of alterations in the eno 5′ UTR on the regulation of eno - cat expression. Top: schematic representation of the region encompassing the eno and pyrG genes in W3110 WT and W3110 PBAD eno strains. Bottom: degree of chloramphenicol resistance of W3110 and W3110 PBAD eno strains harbouring pERS1. The W3110 and W3110 PBAD eno strains harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown to an OD 600 of 0.6 in LB containing 1 mM IPTG and 0.01% arabinose, diluted, and spotted on LB agar containing 0.01% arabinose and 0 (Cm 0) or 100 (Cm 100) μg ml −1 chloramphenicol. (c) Schematic representation of pERS1-derived plasmids that additionally contain a DNA segment encompassing the eno and pyrG genes (from + 875 of the pyrG coding region to + 748, + 320, or + 200 of the eno coding region). (d) Minimal inhibitory concentrations (MICs) of MG1655 harbouring pERS1, pERS-AS748, pERS-AS320, or pERS-AS200 against chloramphenicol. Measurements of the MICs were performed independently, in triplicate, in LB containing various chloramphenicol concentrations; significant differences are indicated with different letters (one-way analysis of variance [ANOVA] followed by the Student–Newman–Keuls test, P
Article Snippet: An aliquot (4 pmol) of 32 P-5′ end labelled RNA was incubated with 1 ng of purified RNase III with 0.25 mg ml−1 yeast tRNA (Thermo Fisher Scientific), 20 U of RNase inhibitor (Takara), and RNase III cleavage buffer, with or without MgCl2 .
Techniques: Expressing, RNA Expression, Transformation Assay, Derivative Assay