Journal: Nucleic Acids Research

Article Title: ProQ-associated small RNAs control motility in Vibrio cholerae

doi: 10.1093/nar/gkae1283

Figure Lengend Snippet: FlaX inhibits ribosome binding in the 5′-UTR of the flaB mRNA. ( A ) The secondary structure of 5′-UTR flaB + first 60 nucleotides with FlaX binding sites highlighted in blue. Deletion of the different binding sites are marked in red. Residues that were substituted with cytosines (see panel B) are indicated by a red ‘C’ placed adjacent to each modified position. The start codon and the SD sequence are marked in orange. ( B ) Translational GFP reporter fusions with deletion mutations in each of the binding sites (as indicated in ( A )) were transformed into E. coli Top10 cells. GFP production was measured, with fluorophore levels from the control strain normalized to 1. Error bars represent the standard deviation of three biological replicates. Statistical significance was determined using ordinary one-way ANOVA with Dunnett’s multiple comparison (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). ( C and D ) In vitro translation of the translational reporter fusion flaB :: gfp mRNA. The protein was monitored by western blot at the indicated time points. Translation of single (C), double and triple mutants (D) (indicated in Figure – ) was measured in the presence or absence of FlaX. Data were collected from two biological replicates. Toeprint assay on flaB mRNA (0.2 pmol) in the presence or absence of FlaX. The presence of the 30S ribosomal subunit, the tRNA fMet and the flaB mRNA is indicated. Wild-type and mutant flaX are added in different ratios relative to the flaB mRNA (0.2 pmol, 0.6 pmol, 1 pmol and 2 pmol for flaX WT and 0.2 pmol and 0.6 pmol for flaX M1 + 2 + 3). Position of the 30S initiation complex is indicated, +15, relative to the start codon.

Article Snippet: Subsequently, 20 pmol of tRNA fMet (MP Biomedicals, 199 154) was introduced, and the formation of the 30S initiation complex (30S-IC) was allowed for 15 min at 37°C. cDNA synthesis was then carried out by adding SuperScript™ II Reverse Transcriptase (Thermo, 18 064 014) and incubating the mixture at 37°C for 20 min.

Techniques: Binding Assay, Modification, Sequencing, Transformation Assay, Control, Standard Deviation, Comparison, In Vitro, Western Blot, Mutagenesis