Rescue of Escherichia coli auxotrophy by de novo small proteins
- Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden
- Department of Cell and Molecular Biology, Uppsala University, Sweden
Figures
Figure 1 with 5 supplements
Experimental setup and sequence characteristics of the isolated small proteins.
(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 …
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Figure 1—source data 1
Random sequence libraries cloned into the expression vector pRD2.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Growth rates of strains expressing Hdps relative to empty pRD2 control.
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. …
Figure 1—figure supplement 2
Characterization of Hdp1 mutations and truncations.
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 …
Figure 1—figure supplement 3
Characterization of Hdp2 mutations and truncations.
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 …
Figure 1—figure supplement 4
Hdp structure predictions using JPred4.
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 …
Figure 1—figure supplement 5
Hdp complementation of various deletion strains.
(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, …
Figure 2 with 2 supplements
Overview of his operon regulation in E. coli and regulatory activity of the Hdps.
(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, …
Figure 2—figure supplement 1
Hdp regulatory activity is independent of the stringent response.
(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 …
Figure 2—figure supplement 2
Protein abundance of strains expressing Hdps relative to the empty plasmid control strain.
‘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 …
Figure 3 with 4 supplements
Characterization of Hdp1opt binding interactions with the his operator mRNA.
(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 …
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Figure 3—source data 1
Source data for Figure 3B and D.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-data1-v2.zip
Figure 3—figure supplement 1
Characterization of Hdp1opt, the Hdp1 variant optimized for increased water solubility.
(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 …
Figure 3—figure supplement 2
Western blot detection of HA-tagged Hdp1 variants following co-immunoprecipitation assays.
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, …
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Figure 3—figure supplement 2—source data 1
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data1-v2.zip
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Figure 3—figure supplement 2—source data 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 1.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data2-v2.zip
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Figure 3—figure supplement 2—source data 3
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data3-v2.zip
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Figure 3—figure supplement 2—source data 4
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data4-v2.zip
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Figure 3—figure supplement 2—source data 5
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data5-v2.zip
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Figure 3—figure supplement 2—source data 6
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp2-data6-v2.zip
Figure 3—figure supplement 3
Electrophoretic mobility shift assay (EMSA) gels and binding curves from fitting of different binding models to the EMSA data.
(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 …
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Figure 3—figure supplement 3—source data 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 replicate 1.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data1-v2.zip
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Figure 3—figure supplement 3—source data 2
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data2-v2.zip
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Figure 3—figure supplement 3—source data 3
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data3-v2.zip
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Figure 3—figure supplement 3—source data 4
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data4-v2.zip
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Figure 3—figure supplement 3—source data 5
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data5-v2.zip
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Figure 3—figure supplement 3—source data 6
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp3-data6-v2.zip
Figure 3—figure supplement 4
RNase T1 probing experimental replicates.
(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 …
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Figure 3—figure supplement 4—source data 1
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-fig3-figsupp4-data1-v2.zip
Additional files
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Supplementary file 1
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.
- https://cdn.elifesciences.org/articles/78299/elife-78299-supp1-v2.xlsx
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MDAR checklist
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