Research Article

Chromosomes and Gene Expression

Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

Chinese Academy of Sciences, China
University of Chinese Academy of Sciences, China
ShanghaiTech University, China
Zhejiang University School of Medicine, China
Chinese National Human Genome Center at Shanghai, China
The Chinese University of Hong Kong, Prince of Wales Hospital, China
Fudan University, China

Dec 17, 2019

https://doi.org/10.7554/eLife.50928

Open access
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Figures
Tables
Additional files

4 figures, 1 table and 4 additional files

Figures

Figure 1 with 2 supplements

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The overall structure of *E. coli* Crl–TAC.

(A) The scaffold used in structure determination of *E. coli* Crl-TAC. (**B, C**) The top and front view orientations of the cryo-EM density map (B) and structure model (C) of *E. coli* Crl–TAC. The RNAP, Crl and nucleic acids are presented as cartoon and colored as indicated in the color key. The density map is shown in gray envelop. (D) The cryo-EM density map (blue transparent surface) for Crl. (E) The cryo-EM density map (red transparent surface) for the upstream junction of the transcription bubble of promoter DNA and σ^S. NT, non-template-strand promoter DNA; T, template-strand promoter DNA.

Figure 1—figure supplement 1

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The preparation of *E. coli* Crl–TAC complex.

(A) Elution peaks of *E. coli* Crl–TAC from a size-exclusion column. (B) SDS-PAGE and (C) native-PAGE of the *E. coli* Crl–AC. The gel was first stained with SYBR Gold for nucleic acids and then with Coomassie Brilliant Blue for proteins.

Figure 1—figure supplement 2

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The processing pipeline for cryo-EM map construction of *E. coli* Crl–TAC.

(A) The flowchart of data processing for the cryo-EM structure of *E. coli* Crl–TAC. (B) Representative cryo-EM micrograph. (C) Representative 2D classifications of *E. coli* Crl–TAC single particles. (D) Angular distribution of *E. coli* Crl–TAC particle projections. (E) The gold-standard Fourier shell correlation (FSC) of *E. coli* Crl–TAC. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 3.80 Å.

Figure 2 with 2 supplements

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The interactions between Crl and the RNAP-holoenzyme.

(A) Crl binds to the σ^S₂ and the RNAP-β’ subunit. Crl is represented as a blue transparent surface and cartoon. (B) σ^S is embedded into a shallow hydrophobic groove of Crl. The electrostatic potential surface of Crl was generated using APBS tools in Pymol. (C) The detailed salt-bridge interaction between the ‘R’ loop of Crl and the ‘specificity loop’ of σ^S. Salt-bridge bonds are shown as blue dashed lines. (D) The N-terminal tail of Crl makes potential weak interactions with RNAP-β’ subunit, probably through R11 and S10. The colors are as in Figure 1. (E) The yeast two-hybrid assay reveals the key interface residues (red) of Crl (left) or σ^S (right). Mutating the key residues disrupts interactions between Crl and σ^S. AD, the activation domain of GAL4; BD, the DNA-binding domain of GAL4. (F) The in vitro transcription assay shows that mutating most of the key interface residues of Crl (left) substantially impairs its transcription activation activity, and that mutating most of the key interface residues of σ^S resulted in reduced response to Crl. ΔR-loop, replacing residue 43–51 with a ‘GSGS’ linker. (G) Protein sequence alignment of Crl from 72 non-redundant bacterial species. Filled circles indicate residues that are involved with interactions with σ^S; filled red circles indicate key contact residues. The residues are numbers as in *E. coli* Crl. NT, non-template-strand promoter DNA; T, template-strand promoter DNA.

Figure 2—figure supplement 1

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The detailed interactions between Crl and σ^S₂.

(A) Structure superimposition of Crl proteins from crystal structure of Crl (PDB: 4Q11; cyan) and Crl in *E. coli* Crl-TAC (blue). (B) The σ^S₂ inserts into a hydrophobic groove on surface of Crl. (C) The interaction between the ‘R’ loop of Crl and the ‘specificity’ loop of σ^S₂. The cryo-EM density maps for the side chains of the interface residues of Crl and σ^S₂ are shown as in blue and green mesh, respectively. The C, N, and O atoms of Crl residues are shown in white, blue, and red, respectively. The C, N, and O atoms of σ^S residues are shown in yellow, blue, and red, respectively. (D) Structure superimposition of our *E. coli* Crl–σ^S–RPo complex and *E. coli* σ^S–RPo complex (PDB 5IPM) reveals similar conformation of the specificity loop. (E) *E. coli* Crl interacts with σ^S₂ in our structure. (F) *Mycobacterium smegmatis* RbpA interacts with σ^A₂ in PDB 5TW1. (G) *Caulobacter cresentus* GcrA interacts with σ⁷⁰₂ in PDB 5YIX. The conserved region and the non-conserved region (NCR) of σ^S₂ in (E), (F), and (G) are shown in yellow and orange, respectively.

Figure 2—figure supplement 2

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The sequence alignment of bacterial σ factors.

(A) Sequence alignment of six σ⁷⁰-type *E. coli* σ factors. (B) The sequence alignment of the σ⁷⁰subunit of ten representative bacterial species (*Proteus mirabilis*, *Escherichia coli*, *Salmonella enterica* serovar Typhi, *Yersinia enterocolitica*, *Shigella flexneri*, *Vibrio cholerae*, *Dickeya dadantii*, *Providencia stuartii*, *Xenorhabdus bovienii*, and *Photobacterium profundum*). (C) The sequence alignment of the σ^S subunit of the same ten bacterial species. The residues are numbered as in *E. coli*.

Figure 3 with 4 supplements

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Crl promotes assembly of the σ^S-RNAP holoenzyme by stabilizing σ^S₂.

(A) Crl increases binding affinity between RNAP and σ^S in a fluorescence polarization (FP) assay. (B) Deletion the ‘R’ loop but not the N-tail (ΔN-tail; deleting residue 1–11) abolishes the ability of Crl to promote the assembly of the σ^S-RNAP holoenzyme. (C) Hydrogen-to-deuterium exchange (HDX) profile of free σ^S after 10 s exposure to deuteron. Incorporation was mapped onto the cryo-EM structure of σ^S. The inset shows the color coding for different percentages of deuteron incorporation. (D) HDX changes for σ^S in the presence of Crl as compared to free σ^S, mapped on the cryo-EM structure of σ^S. The percentage difference in deuterium uptake values represents an average across all four time points, ranging from 10 s to 900 s. For heatmap color coding, pink, cyan, and gray indicate increase, decrease, and no significant change in HDX, respectively. Dark grey represents regions that were not consistently resolved in all HDX experiments. (E) Deuterium uptake plots and mass spectra of indicated peptides from helices α4 and α5 in the absence and presence of Crl. Left: the deuterium uptake data are plotted as percent deuterium uptake versus time on a logarithmic scale. Right: mass spectra of the indicated peptides at different labeling time points, with the mass spectra of undeuterated samples shown as controls. (F) Peak-width analysis at different time points for peptides from helices α4 and α5 reveal the existence of EX1 kinetics. (**C–F**) The HDX experiments were repeated at least twice. (G) σ^S₂ regions with reduced HDX rate upon Crl binding, the colors are as in (D).

Figure 3—source data 1 The raw data of Figure 3A-B.: https://cdn.elifesciences.org/articles/50928/elife-50928-fig3-data1-v1.xlsx
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Figure 3—figure supplement 1

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HDX profile of the σ^S protein in the absence of Crl.

(A) The relative deuteration levels (%D) of each peptide were calculated with the assumption that a fully deuterated sample retains 90% D in the current LC setting and plotted as a function of peptide position. The blue to red lines represent data acquired at 10 s up to 900 s deuteration. (B) Heat map of σ^S protein at different time points. The inset shows the color coding for different percentages of deuteron incorporation.

Figure 3—figure supplement 2

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Representative peptides from helix α2, α3, α4 and α5 of σ^S₂ whose HDX rate decreased considerably in the presence of Crl.

(**A–D**) Deuterium uptake plots, peak width plots and mass spectra of indicated peptides from σ^S₂ in the absence and presence of Crl. Left: the deuterium uptake data are plotted as percent deuterium uptake versus time on a logarithmic scale. Middle: mass spectra of indicated peptides at different labeling time points, with the mass spectra of undeuterated samples shown as controls. Right: peak-width analysis at different time points for peptides covering helices α2–α5.

Figure 3—figure supplement 3

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Deuterium uptake plots for additional peptides from σ^S whose HDX rate did not change in the presence of Crl.

The deuterium uptake data are plotted as percent deuterium uptake versus time on a logarithmic scale.

Figure 3—figure supplement 4

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Peptide coverage of σ^S.

The sequence coverage map for σ^s in the HDX-MS study. Green lines shown below the protein sequence represent the digested peptides identified and analyzed in this study.

Figure 4 with 1 supplement

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Crl facilitates RPo formation.

(A) Crl increases transcription by a pre-assembled σ^S-RNAP holoenzyme from the PosmY promoter in a concentration-dependent manner. (B) Crl promotes σ^S-RPo formation in a fluorescence stopped-flow experiment using Cy3-labeled λP_R promoter DNA. (C) Crl promotes σ^S-RPo formation in a fluorescence stopped-flow experiment using 2AP-labeled λP_R promoter DNA. (D) The KMO₄ footprinting experiment result shows that Crl increases formation of the RPo complex. The *osmY* promoter dsDNA with ³²P-labeled at the 5′-end of the non-template strand was used for the assay. The hyperactive T-stretch (−9 to −7) in the transcription bubble upon RPo formation is highlighted in red and labeled on the right. (E) The upstream promoter DNA is unwound by the W-dyad (W148 and W149), and the unwound A_-11 nucleotide of the non-template strand DNA is recognized and stabilized by a protein pocket on σ^S₂. (F) The ‘R’ loop of Crl stabilizes the conformation of the ‘specificity’ loop that forms the pocket for unwinding and recognizing the A_-11 nucleotide of non-template strand promoter DNA.

Figure 4—figure supplement 1

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The promoter DNA used for stopped-flow fluorescence experiments.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Escherichia coli)	crl		Gene bank (7152411)
Strain, strain background (E. coli)	BL21(DE3)	Novo protein, China	V211-01A	Chemically competent cells
Recombinant DNA reagent	pET-28a-TEV-Crl (plasmid)	This paper		Protein expression vector for E. coli Crl
Recombinant DNA reagent	pET-28a-TEV-Ecs^S (plasmid)	This paper		Protein expression vector for E. coli s^S
Recombinant DNA reagent	pET-28a-TEV-Crl (P21W) (plasmid)	This paper		Protein expression vector for E. coli Crl (P21W)
Recombinant DNA reagent	pET-28a-TEV-Crl (Y22A) (plasmid)	This paper		Protein expression vector for E. coli Crl (Y22A)
Recombinant DNA reagent	pET-28a-TEV-Crl (R24A) (plasmid)	This paper		Protein expression vector for E. coli Crl (R24A)
Recombinant DNA reagent	pET-28a-TEV-Crl (L38A) (plasmid)	This paper		Protein expression vector for E. coli Crl (L38A)
Recombinant DNA reagent	pET-28a-TEV-Crl (R51A) (plasmid)	This paper		Protein expression vector for E. coli Crl (R51A)
Recombinant DNA reagent	pET-28a-TEV-Crl (F53A) (plasmid)	This paper		Protein expression vector for E. coli Crl (F53A)
Recombinant DNA reagent	pET-28a-TEV-Crl (F76A) (plasmid)	This paper		Protein expression vector for E. coli Crl (F76A)
Recombinant DNA reagent	pET-28a-TEV-Crl (ΔN-tail) (plasmid)	This paper		Protein expression vector for E. coli Crl (Δ1–11)
Recombinant DNA reagent	pET-28a-TEV-Crl (ΔR-loop) (plasmid)	This paper		pET-28a-TEV-Crl (ΔR-loop; residues 43–51 of Crl by a ‘GSGS’ linker)
Recombinant DNA reagent	pET-28a-TEV-Crl (ΔN-tail /ΔR-loop) (plasmid)	This paper		Protein expression vector for E. coli Crl (ΔN-tail /ΔR-loop)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S(R81A) (plasmid)	This paper		Protein expression vector for E. coli σ^S (R81A)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S (R82A) (plasmid)	This paper		Protein expression vector for E. coli σ^S (R82A)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S (R85A) (plasmid)	This paper		Protein expression vector for E. coli σ^S (R85A)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S (D135A) (plasmid)	This paper		Protein expression vector for E. coli σ^S (D135A)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S (E137A) (plasmid)	This paper		Protein expression vector for E. coliσ^S (E137A)
Recombinant DNA reagent	pET-28a-TEV-Ecσ^S (A239C) (plasmid)	This paper		Protein expression vector for E. coli σ^S (A239C)
Recombinant DNA reagent	pGADT7-Crl (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (P21W) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (P21W) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (Y22A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (Y22A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (R24A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (R24A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (E25A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (E25A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (L38A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (L38A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (V44A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (V44A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (K45A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (K45A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (R51A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (R51A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (F53A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (F53A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (F76A) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (F76A) is fused to Gal4-AD
Recombinant DNA reagent	pGADT7-Crl (ΔR-loop) (plasmid)	This paper		Plasmid for yeast two-hybrid; Crl (ΔR-loop) is fused to Gal4-AD
Recombinant DNA reagent	pGBKT7-σ^S₂ (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂(53–162) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (Y78A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (Y78A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (F79A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂(F79A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (R81A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (R81A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (R82A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (R82A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (R85A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (R85A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (R93A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (R93A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (D135A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (D135A) is fused to Gal4-BD
Recombinant DNA reagent	pGBKT7-σ^S₂ (E137A) (plasmid)	This paper		Plasmid for yeast two-hybrid; σ^S₂ (E137A) is fused to Gal4-BD
Recombinant DNA reagent	pEasyT-PosmY (plasmid)	This paper		Plasmid containing PosmY
Commercial assay or kit	Ezmax one-step cloning kit	Tolo Bio-tech, China	Cat#24305–01
Other	Ni-NTA agarose	smart-lifesciences, China	Cat#SA004100
Other	C-flat CF-1.2/1.3 400 mesh	Electron Microscopy Sciences	Cat#CF413-100