Structure buffer

The sRNAs AbcR1 WT, QC1, QC2, and QC1+2 and the target mRNA fragments (comprising ~150 nucleotides in the TIR or in the CDS) were synthesized in vitro by runoff transcription with T7 RNA polymerase from the linearized plasmids listed in Table S4. 5‘ end labeling of AbcR1 WT or AbcR1 variants (QC1, QC2, and QC1+2) with ³²P was performed as described.¹⁰² (link) RNA band shift experiments were performed in 1x structure buffer (Ambion) in a total reaction mixture volume of 15 µl as follows. 5′ end labeled AbcR1 (corresponding to 5000 c.p.m.) and 1 µg of tRNA were incubated in the presence of unlabeled target mRNA fragments (~150 nt) at 30 °C for 20 min. The final concentrations of added unlabeled RNA fragments are given in the figure legends. Prior to gel loading, the binding reactions were mixed 4.5 µl of native loading dye (50% glycerol, 0.5× TBE, 0.1% bromophenol blue and 0.1% xylene cyanol) and run on native 6% polyacrylamide gels in 0.5× TBE buffer at 300 V for 1.5–3 h.

For RNA in vitro synthesis, ∼200 ng of template DNA amplified from Salmonella genomic DNA using JVO-7023/JVO-7025 (SdsR) and JVO-7024/JVO-7025 (SdsR +31) were reverse transcribed employing the MEGAscript kit (Ambion) according to the manufacturer's guidelines. Correct size and integrity of the RNA were confirmed on a denaturing polyacrylamide gel. Formation of complexes between sRNAs and Hfq in vitro was analyzed by EMSA as described previously (43 (link)). Briefly, 5′ end-labelled RNA (4 pmol) was denatured (95°C, 2 min), chilled on ice for 5 min and supplemented with 1× structure buffer (Ambion) and 1 μg yeast RNA. Upon addition of purified Hfq (lab stock; concentration as indicated in the figure legend) or Hfq dilution buffer (control), samples were incubated at 37°C for 10 min. Prior to loading, reactions were mixed with native loading buffer, and separated by native PAGE. Gels were dried and signals were determined on a Typhoon FLA 7000 phosphorimager (GE Healthcare).

Templates for in vitro transcription by T7 RNA polymerase (New England Biolabs; #M0251) of SorY (86nt), PcrZ (136nt), the takP mRNA (92nt) and the RSP_2591 (177nt) mRNA (both fragments containing 5′ UTR and the beginning of the coding sequence) were generated from corresponding primer pairs listed in Table S4. [α-³²P]-UTP (Hartmann Analytic; FP-210) was used to label takP transcripts internally. In vitro transcripts were treated with DNaseI (Promega; M6101) prior to gel purification. RNA band shift experiments were performed in 1× structure buffer (Ambion) in a total reaction mixture volume of 10 µl as follows. Transcripts were heated (90°C; 10 min), cooled down to 4°C and renatured at 32°C separately. 150 fmol of labeled takP RNA was incubated at 32°C for 20 min with unlabeled SorY (ratios 1:1, 1:10, 1:100) or as controls with water, unspecific sRNA (PcrZ) and mRNA (RSP_2591) in 100 fold molar excess. Binding reactions were mixed with 3 µl of native loading dye (50% glycerol, 0.5× TBE, 0.1% bromophenol blue) and ran on native 6% polyacrylamide gels in 0.25× TBE buffer at 150 V for 2 h.