(A) Libraries cloned into the expression vector pRD2. (B) Plasmid transformation into auxotrophic mutants and selection for rescue of auxotrophic mutants. (C) Hydropathy profiles of the three …
Random sequence libraries cloned into the expression vector pRD2.
Relative exponential phase growth rates of E. coli strains expressing various Hdp constructs in either the (A) ∆sierB PhisL-hisoperator-lacZ reporter or (B) MG1655 wild-type genetic background. …
Growth of the E. coli ∆serB auxotrophic strain carrying either the empty pRD2 plasmid or pRD2 containing various mutations or truncations to the Hdp1 coding sequence on M9 minimal medium …
Growth of the E. coli ∆serB auxotrophic strain carrying either the empty pRD2 plasmid or pRD2 containing various mutations or truncations to the Hdp2 coding sequence on M9 minimal medium …
The consensus prediction of Hdp1-3 based on the secondary structure prediction server JPred4. Predicted helices are indicated with red and sheets are marked as green arrows. Confidence values …
(A) Growth of various E. coli deletion strains carrying either the empty pRD2 plasmid or pRD2 encoding Hdp1-3, SerB, or HisB on M9 minimal medium supplemented with 50 µg/ml ampicillin, 0.2% glucose, …
(A) (i) The regulatory region and structural genes of the his operon, including hisB (highlighted in dark gray), (ii) the his operator and RNA secondary structure under histidine-rich conditions, …
(A) β-Galactosidase activity (in Miller Units) of the ∆serB strain carrying a lacZ transcriptional fusion under the control of the rrnB P1 promoter and accompanying regulatory sequence upon …
‘Volcano plots’ showing significant changes in protein abundance in the ∆serB mutant containing the full-length PhisL-hisoperator-lacZ reporter upon expression of Hdp2 and Hdp3, versus the same …
(A) Enrichment of select RNA transcripts in the HA-tagged Hdp1opt and Hdp1opt L27Q mutant pull-down samples, as quantified by RT-qPCR. thrL and thrA are nonspecific control RNAs. RT-qPCR data from …
Source data for Figure 3B and D.
(A) Amino acid changes that generated the Hdp1opt variant with improved water solubility. Hdp1opt(+cys) is a prototype Hdp1opt variant that retains its cysteine residues. Colors represent the …
Protein fractions of the different steps of the co-immunoprecipitation experiments were separated on 16.5% Tris-Tricine polyacrylamide gels and the HA-tagged proteins of interest (~7.7 kDa, …
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
HA-tagged proteins of interested were detected using HRP-conjugated anti-HA mouse monoclonal antibody and Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and visualized using a Bio-Rad ChemiDoc MP System (Chemi Hi Sensitivity setting). Uncropped membrane from experimental replicate 1.
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
White light image of membrane to show protein ladders for size reference. Uncropped membrane from experimental replicate 1.
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
Merged image of membranes from Figure 3—figure supplement 2—source data 1 and Figure 3—figure supplement 2—source data 2 to show protein ladders for size reference alongside detected proteins of interest. Uncropped membrane from experimental replicate 1; included in Figure 3—figure supplement 2 as a representative western blot for the pull-down assays.
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
HA-tagged proteins of interested were detected using HRP-conjugated anti-HA mouse monoclonal antibody and Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and visualized using a Bio-Rad ChemiDoc MP System (Chemi Hi Sensitivity setting). Uncropped membrane from experimental replicate 2.
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
White light image of membrane to show protein ladders for size reference. Uncropped membrane from experimental replicate 2.
Western blot containing protein fractions from the different steps of the co-immunoprecipitation experiments.
Merged image of membranes from Figure 3—figure supplement 2—source data 4 and Figure 3—figure supplement 2—source data 5 to show protein ladders for size reference alongside detected proteins of interest. Uncropped membrane from experimental replicate 2.
(A) Electrophoretic mobility shift assays (EMSAs) of the full-length (A) his operator RNA or (B) thr operator RNA in the presence of increasing concentrations (0–5.5 μM) of Hdp1opt or the Hdp1opt …
Electrophoretic mobility shift assay (EMSA) of the full-length his operator RNA in the presence of increasing concentrations of Hdp1opt.
Uncropped gel from experimental replicate 1.
Electrophoretic mobility shift assay (EMSA) of the full-length his operator RNA in the presence of increasing concentrations of Hdp1opt.
Uncropped gel from experimental replicates 2 and 3. Experimental replicate 2 is included in the main text figure as a representative EMSA gel image.
Electrophoretic mobility shift assay (EMSA) of the full-length his operator RNA in the presence of increasing concentrations of Hdp1opt (left) or the Hdp1opt L27Q mutant (right).
Uncropped gel from Hdp1opt experimental replicate 4 and Hdp1opt L27Q experimental replicate 1. Hdp1opt L27Q experimental replicate 1 is included in Figure 3—figure supplement 3 as a representative EMSA gel image.
Electrophoretic mobility shift assay (EMSA) of the full-length his operator RNA in the presence of increasing concentrations of the Hdp1opt L27Q mutant.
Uncropped gel from experimental replicates 2 and 3.
Electrophoretic mobility shift assay (EMSA) of the full-length thr operator RNA in the presence of increasing concentrations of Hdp1opt.
Uncropped gel from experimental replicate 1; included in Figure 3—figure supplement 3 as a representative EMSA gel image. Unlabeled experiment on the left of the gel was not used for quantification or analysis due to the gel ripping.
Electrophoretic mobility shift assay (EMSA) of the full-length thr operator RNA in the presence of increasing concentrations of Hdp1opt.
Uncropped gel from experimental replicates 2 and 3. ‘X’ denotes a well in which a reaction was loaded in the incorrect order.
(A) RNase T1 probing gels for the full-length his operator RNA in the absence and presence of Hdp1opt (0, 0.69, and 5.5 μM). NR denotes RNA subject to no reaction, OH indicates partial alkaline …
Uncropped RNase T1 probing gel for the full-length his operator RNA in the absence and presence of Hdp1opt (0, 0.69, and 5.5 μM).
NR denotes RNA subject to no reaction, OH indicates partial alkaline hydrolysis, and T1 is an RNase T1 digest of the RNA under denaturing conditions used to map the RNA sequence. (i) Probing reactions incubated with 0.05U T1 RNase for 5 min. (ii) Probing reactions incubated with 0.01U T1 RNase for 5 min. (iii) Probing reactions incubated with 0.05U T1 RNase for 10 min. Numbering of G nucleotides is shown on the left. Arrows highlight changes in RNA cleavage in the presence of Hdp1opt: black arrows indicate nucleotides with increased cleavage and white arrows indicate reduced cleavage. Sequence and/or structure characteristics of the his operator RNA are also indicated on the right (i.e., the Shine-Dalgarno sequence [SD], start codon [AUG], and stop codon [UAG] of the hisL leader peptide coding sequence). The reactions from (iii) are included in the main text figure as a representative T1 RNase probing gel image; all replicates are in included in Figure 3—figure supplement 4. All reaction sets were performed with independent RNA and protein dilutions.
Supplementary Tables 1a-g.
(a) KEIO deletion strains screened with the random sequence libraries. (b) Summary of Hdp1-2 single mutants obtained via random mutagenesis. (c) Escherichia coli K-12 strains used in this study. (d) Fraction bound data as quantified and calculated from the electrophoretic mobility shift assay (EMSA) gels. (e) Parameters from fitting of different binding models to the EMSA data. (f) Plasmids used in this study. (g) Oligonucleotides used in this study.