Jing Shi’s research while affiliated with Nanjing University of Chinese Medicine and other places

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Publications (30)


Fig. 2 C binds promoter DNA as a centrosymmetric tetramer in the phage Mu C-TAC. 751 (A) Relative locations of the centrosymmetric C tetramer at the upstream double-stranded DNA 752 (left panel); structure of C tetramer in cartoon (right panel). (B) The relative locations between 753 C I and C II (left panel) or C III and C IV (right panel) bound to the promoter DNA. The secondary 754 structural elements involved in C tetramer are labeled, respectively. Colors of C I , C II , C III , C IV , 755 NT, and T in A-C are shown as in Fig. 1C. (C) Detailed dimerization interactions between C I 756 and C II (left panel) or C III and C IV (right panel). (D) Detailed tetrameric interactions between C I 757 and C IV or C II and C III . The secondary structural elements are labeled, respectively. Colors are 758
Fig. 3 C makes extensive interactions with the conserved domains of RNAP in the phage 765 Mu C-TAC. 766 (A) Relative locations of E. coli RNAP αCTD, C I , and the upstream double-stranded DNA. E. 767
Fig. 5 Proposed transcription activation model for the phage Mu Mor/C family proteins. 818 (A) The interface of σ 70 R4 with Mor (left panel) or C (middle panel), Mor I or C I and σ 70 R4
Figures and Figure Legends 736 737
Structural insight into the transcription activation mechanism of the phage Mor/C family activators
  • Preprint
  • File available

May 2025

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15 Reads

Jing Shi

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Zonghang Ye

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Yirong Huang

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[...]

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Wei Lin

Bacteriophage Mu, a temperate phage that infects E. coli K-12 and other enteric bacteria, precisely controls its replication cycle through hijacking host RNA polymerase (RNAP) by the middle operon regulator Mor and the late gene transcription activator C. Though a dimeric arrangement and significant conformational changes are proposed for the distinct Mor/C family activators, the underlying transcription activation mechanism remains unclear. In this study, we present two cryo-EM structures of the transcription activation complex (Mor-TAC and C-TAC) with phage Mu middle and late gene promoters, respectively. Remarkably, the Mor/C activators bind to promoter DNA as a centrosymmetric tetramer rather than as the proposed dimer, concurrently stabilizing by the N-terminal dimerization domains and C-termini. The C-terminal DNA binding domains and two anti-β-strands simultaneously interact with two adjacent DNA major grooves. The activators also engage a variety of interactions with the conserved domains (αCTD, σ70R4, and β FTH) of RNAP, providing evidences for a recruitment mechanism. In addition, single-molecule FRET assays show that C significantly enhances RPitc formation, suggesting a different multi-step activation mechanism for C. Collectively, these findings reveal the unique transcription activation mechanism of tetrameric Mor/C family activators, unraveling a novel mode of phage hijacking and bacterial transcription regulation.

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The overall structures of Mtb PhoP-TAC
a DNA scaffold used in structure determination of Mtb 2PhoP-TAC (top panel) and two views of the cryo-EM density map of Mtb 2PhoP-TAC (bottom panel). b DNA scaffold used in structure determination of Mtb 4PhoP-TAC (top panel) and two views of the cryo-EM density map of Mtb 4PhoP-TAC (bottom panel). c DNA scaffold used in structure determination of Mtb 6PhoP-TAC (top panel) and two views of the cryo-EM density map of Mtb 6PhoP-TAC (bottom panel). According to the structures, the PhoP binding box, −35 element, -10 element is boxed with yellow, violet, and brown colors, respectively. a1 site, b1 site, a2 site, b2 site, a3 site and b3 site of the PhoP binding box are shaded with light orange, dark cyan, brown, cyan, purple, and dark green, respectively. The EM density maps are colored as indicated in the color key. NT, non-template-strand promoter DNA; T, template-strand promoter DNA.
Tandem PhoP hexamer engages promoter DNA in Mtb 6PhoP-TAC
a Relative locations of Mtb PhoPI_DBD, PhoPII_DBD, PhoPIII_DBD, PhoPIV_DBD, PhoPV_DBD, PhoPVI_DBD located at the upstream double-stranded DNA. b, The relative locations between PhoPI_DBD and PhoPII_DBD bound to the promoter DNA. The secondary structural elements involved in PhoPI_DBD and PhoPII_DBD are labeled, respectively. c Detailed interactions between Mtb PhoPI_DBD, PhoPII_DBD, and their corresponding PhoP binding sites. The key residues involved are shown as green spheres. d The detailed interactions between PhoPI_DBD and PhoPII_DBD. The key residues involved in PhoPI_DBD and PhoPII_DBD are shown as wheat and blue spheres, respectively. e Substitutions of PhoP residues involved in promoter engagement and dimeric interface reduce in vitro transcription activities. Data for in vitro transcription assays are means of 3 technical replicates. Error bars represent mean ± SEM of n = 3 experiments. Source data are provided as a Source Data file. f Relative locations of PhoPII_DBD, σAR4, and the upstream double-stranded DNA in Mtb 6PhoP-TAC. g Relative locations of σAR4 and the upstream typical −35 element DNA in Mtb RPo (PDB ID: 6VVY). Colors are shown as in Fig. 1c.
The critical protein–protein interactions between Mtb PhoP and the conserved β flap and σAR4 domains of RNAP in Mtb PhoP-TACs
a Relative locations of Mtb PhoPI_DBD and RNAP β flap (Left and Middle). PhoPI_DBD and RNAP β flap are shown in surface style (Left). Residues involved between PhoPI_DBD and RNAP β flap are shown as slate (PhoPI_DBD) and cyan (RNAP β flap) spheres (Right). b Relative locations of Mtb PhoP_DBDs and RNAP σAR4 (Left and Middle). PhoPI_DBD, PhoPII_DBD and RNAP σAR4 are shown in surface (Left). Residues involved in interactions between PhoP_DBDs and RNAP σAR4 are shown as blue (PhoPI_DBD), wheat (PhoPII_DBD) and yellow (RNAP σAR4) spheres (Middle and Right). RNAP β flap is colored in cyan, and RNAP σAR4 is colored in yellow. c Conformational comparisons of αCTD and DNA from 6PhoP-TAC and GlnR-TAC. gray, PhoP or GlnR; pink, αCTD; red, DNA from 6PhoP-TAC; gray, DNA from GlnR-TAC. d Substitutions of PhoP residues involved in PhoP-β flap, PhoP-σAR4 interfaces compromise in vitro transcription activities. Data for in vitro transcription assays are means of 3 technical replicates. Error bars represent mean ± SEM of n = 3 experiments. Source data are provided as a Source Data file. The other colors are shown as in Fig. 1.
The cryo-EM map and structural model of Mtb PhoP-TRC
a Gel filtration maps and SDS-PAGE analysis of Mtb PhoP-TRC. Source data are provided as Source Data files. b Relative transcription activity of Mtb PhoP on the PhoP-GlnR co-regulated amtB promoter and the amtB_mut promoter determined by in vitro transcription assays. Mutations of PhoP (truncation of the PhoP_REC domain) or the upstream PhoP binding sites (amtB_mut promoter) cause defects on the transcription repression activity. Data for in vitro transcription assays are means of 3 technical replicates. Error bars represent mean ± SEM of n = 3 experiments. Source data are provided as a Source Data file. c DNA scaffold used in structure determination of Mtb PhoP-TRC. GlnR binding box (a1-b1 sites, a2-b2 sites, and a3-b3 sites) and PhoP binding box (a3’-b3’ sites) are framed in green and yellow color, respectively. The a1 site, b1 site, a2 site, b2 site, a3 site, b3 site, a3’ site, b3’ site are shaded in light orange, dark cyan, brown, cyan, khaki, light green, purple, and dark green, respectively. d Two views of the cryo-EM density map of Mtb PhoP-TRC. The EM density map is colored as indicated in the color key. e The promoter DNA bends toward the main body of RNAP in the preliminary structure model of PhoP-TRC compared with the modeled structure of Mtb 6GlnR-TAC. Mtb 6GlnR-TAC is modeled based on the structural similarities of 6PhoP-TAC. The other colors are shown as in Fig. 1.
Proposed working model for PhoP-dependent transcription regulation
a The canonical RPo and three types of PhoP-dependent transcription activation in which PhoP exists as a tandem dimer, tetramer, and hexamer, respectively. b Classic transcription repression models by occluding RNAP binding sites at a promoter (upper panel) or locking RNAP on a promoter (lower panel). c A proposed “competitive occluding model” for PhoP-dependent transcription repression. In the absence of the upstream PhoP dimer, six GlnR molecules cooperatively and efficiently activate transcription of the target promoter. However, when the PhoP dimer interacts competitively with the a3’-b3’ sites and engages the adjacent GlnR molecules, these interactions cause significant distortion of the upstream DNA in the long spacer region, creating steric hindrances that impede the binding of a third GlnR dimer. Consequently, the formation and stabilization of a competent 6GlnR-TAC is disrupted, resulting in the repression of efficient GlnR-dependent transcription initiation. The repressor and spacer are represented as a five-pointed star and a rectangle, respectively, both colored in red.
Structural insights into transcription regulation of the global OmpR/PhoB family regulator PhoP from Mycobacterium tuberculosis

February 2025

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31 Reads

As a global transcription activator or repressor, the representative OmpR/PhoB family response regulator PhoP plays a crucial role in regulating bacterial pathogenicity and stress adaptation. However, the molecular mechanisms underlying the transcriptional regulation that define its differential functions remain largely unclear. In the present study, we determine three cryo-EM structures of Mycobacterium tuberculosis (Mtb) PhoP-dependent transcription activation complexes (PhoP-TACs) and build one preliminary cryo-EM structure model of Mtb PhoP-dependent transcription repression complex (PhoP-TRC). In PhoP-TACs, tandem PhoP dimers cooperatively recognize various types of promoters through conserved PhoP-PHO box interactions, which displace the canonical interactions between the -35 element and σAR4 of RNA polymerase (RNAP), unraveling complex transcription activation mechanisms of PhoP. In PhoP-TRC, one PhoP dimer binds and significantly distorts the upstream PHO box of the promoter cross-talked with the global nitrogen regulator GlnR through the PhoP-PHO box, PhoP-GlnR and αCTD-DNA interactions. This unique binding of PhoP creates steric hindrances that prevent additional GlnR binding, positioning PhoP within a unique ‘competitive occluding model’, as supported by prior biochemical observations. Collectively, these findings reveal the dual molecular mechanisms of PhoP-dependent transcription regulation, and offer valuable insights for further exploration of the enormous PhoP-like OmpR/PhoB family response regulators.


Single-molecule reconstruction of eukaryotic factor-dependent transcription termination

June 2024

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98 Reads

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4 Citations

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.


Figure 2. The interactions between AfsR_DBD and promoter DNA (A) Two AfsR_DBDs in complex with the afsS promoter DNA. AfsR I _DBD and AfsR II _DBD are shown as blue or orange cartoon labeled with secondary structures, respectively. (B) Detailed interactions between the AfsR_DBD molecules and promoter DNA. Residues from AfsR_DBD involved in contacting the A site (left panel) and B site (right panel) of AfsR binding box are shown as green spheres. The double strands of DNA are represented as red and dark red sticks, respectively. (C) In vitro transcription assays used for evaluating the substitution effects of AfsR residues involved in promoter recognition. Error bars represent mean G SEM of n = 3 experiments. Significant differences from the wild-type AfsR are analyzed by one-way ANOVA with Dunnett's multiple comparison test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05), respectively.
Figure 3. Three interfaces stabilize the dimeric assessment of AfsR_SARPs in AfsR-RPi (A) The dimeric AfsR_SARPs engage promoter DNA through three interfaces, which is indicated by circle dotted lines and enlarged in (B)-(D), respectively. (B) Interface I: detailed interactions between AfsR I _BTAD and AfsR II _DBD. (C) Interface II: detailed interactions between AfsR I _BTAD and AfsR II _BTAD. (D) Interface III: detailed interactions between AfsR I _BTAD and AfsR II _BTAD. The residues from AfsR II and AfsR I involved in the dimeric interfaces between AfsR I _SARP and AfsR II _SARP are shown as blue and orange spheres, respectively. (E) In vitro transcription assays used for evaluating the substitution effects of AfsR residues involved in the dimeric interfaces of AfsR_SARPs. Error bars represent mean G SEM of n = 3 experiments. Significant differences from the wild-type AfsR are analyzed by one-way ANOVA with Dunnett's multiple comparison test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05), respectively.
Figure 4. The interactions between AfsR_SARPs and the conserved domains of RNAP (A) Relative locations of S. coelicolor AfsR I _DBD and RNAP b flap (left and middle). AfsR I _DBD and RNAP b flap are shown in blue and cyan surface, respectively (left). Residues involved are shown as blue (AfsR I _DBD) and cyan (RNAP b flap) spheres (right). (B) Relative locations of S. coelicolor A3(2) AfsR_SARPs and RNAP s HrdB R4 (left panel). AfsR I _BTAD, AfsR II _DBD, and RNAP s HrdB R4 are shown in surface (left). Residues involved are shown as orange (AfsR II _DBD), blue (AfsR I _BTAD), and yellow (RNAP s HrdB R4) spheres (middle and right). (C) Relative locations of S. coelicolor A3(2) AfsR_SARPs and RNAP aCTD (left). AfsR I _BTAD, AfsR II _BTAD, and RNAP aCTD are shown in surface (left). Residues involved are shown as orange (AfsR II _BTAD), blue (AfsR I _BTAD), and yellow (RNAP aCTD) spheres (middle and right). The conserved domains of RNAP b flap, s HrdB R4, and aCTD are colored in cyan, yellow, and violet, respectively. The protein-protein interfaces enlarged in the middle or/and right panels (A-C) are indicated by circle dotted lines in the corresponding left panels, respectively. (D) In vitro transcription assays used for evaluating the substitution effects of AfsR residues involved in AfsR-b flap, AfsR-s HrdB R4, and AfsR-aCTD interfaces. Error bars represent mean G SEM of n = 3 experiments. Significant differences from the wild-type AfsR are analyzed by one-way ANOVA with Dunnett's multiple comparison test (****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05), respectively. The other colors are shown as in Figure 1.
Figure 5. Proposed model for SARP-dependent transcription activation
Structural insights into transcription activation of the Streptomyces antibiotic regulatory protein, AfsR

June 2024

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40 Reads

iScience

The Streptomyces antibiotic regulatory proteins (SARPs) are ubiquitously distributed transcription activators in Streptomyces and control antibiotics biosynthesis and morphological differentiation. However, the molecular mechanism behind SARP-dependent transcription initiation remains elusive. We here solve the cryo-EM structure of an AfsR-loading RNA polymerase (RNAP)-promoter intermediate complex (AfsR-RPi) including the Streptomyces coelicolor RNAP, a large SARP member AfsR, and its target promoter DNA that retains the upstream portion straight. The structure reveals that one dimeric N-terminal AfsR-SARP domain (AfsR-SARP) specifically engages with the same face of the AfsR-binding sites by the conserved DNA-binding domains (DBDs), replacing σHrdBR4 to bind the suboptimal −35 element, and shortens the spacer between the −10 and −35 elements. Notably, the AfsR-SARPs also recruit RNAP through extensively interacting with its conserved domains (β flap, σHrdBR4, and αCTD). Thus, these macromolecular snapshots support a general model and provide valuable clues for SARP-dependent transcription activation in Streptomyces.


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Structural insights into transcription regulation of the global virulence factor PhoP from Mycobacterium tuberculosis

May 2024

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92 Reads

Mycobacterium tuberculosis (Mtb), remaining as the leading cause of the worldwide threat Tuberculosis, relies heavily on its transcriptional reprogramming of diverse stress genes to swiftly adapt to adverse environments and ensure infections. The global virulence factor PhoP plays a pivotal role in coordinating transcription activation or repression of the essential phosphate-nitrogen metabolic remodeling genes. However, what defines PhoP to deferentially act as an activator or a repressor remains largely unexplored. Here, we determine one cryo-EM structure of Mtb RNAP-promoter open complex, three cryo-EM structures of PhoP-dependent transcription activation complexes (PhoP-TACs) consisting of Mtb RNA polymerase (RNAP), different number of PhoP molecules binding to different types of well-characterized consensus promoters, and one cryo-EM structure of Mtb PhoP-dependent transcription repression complex (PhoP-TRC) comprising of Mtb RNAP, PhoP, the nitrogen metabolism regulator GlnR and their co-regulated promoter. Structural comparisons reveal phosphorylation of PhoP is required for stabilization of PhoP-TACs, PhoP specifically recognizes promoters as novel tandem dimers and recruits RNAP through extensively interacting with its conserved β flap and σAR4 domains. Strikingly, the distinct promoter spacer length and PhoP-GlnR interactions in PhoP-TRC constrain the upstream DNA into a distinct topology and retain PhoP in a novel ‘dragging repression mode’. Collectively, these data highlight the dual regulatory mechanisms of PhoP-dependent transcription regulation in governing stress adaptation. These findings provide structural basis for developing potential anti-tuberculosis drugs and/or interventions.


Structural insights into transcription regulation of the global virulence factor PhoP from Mycobacterium tuberculosis

May 2024

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15 Reads

Mycobacterium tuberculosis (Mtb), remaining as the leading cause of the worldwide threat Tuberculosis, relies heavily on its transcriptional reprogramming of diverse stress genes to swiftly adapt to adverse environments and ensure infections. The global virulence factor PhoP plays a pivotal role in coordinating transcription activation or repression of the essential phosphate-nitrogen metabolic remodeling genes. However, what defines PhoP to deferentially act as an activator or a repressor remains largely unexplored. Here, we determine one cryo-EM structure of Mtb RNAP-promoter open complex, three cryo-EM structures of PhoP-dependent transcription activation complexes (PhoP-TACs) consisting of Mtb RNA polymerase (RNAP), different number of PhoP molecules binding to different types of well-characterized consensus promoters, and one cryo-EM structure of Mtb PhoP-dependent transcription repression complex (PhoP-TRC) comprising of Mtb RNAP, PhoP, the nitrogen metabolism regulator GlnR and their co-regulated promoter. Structural comparisons reveal phosphorylation of PhoP is required for stabilization of PhoP-TACs, PhoP specifically recognizes promoters as novel tandem dimers and recruits RNAP through extensively interacting with its conserved β flap and σAR4 domains. Strikingly, the distinct promoter spacer length and PhoP-GlnR interactions in PhoP-TRC constrain the upstream DNA into a distinct topology and retain PhoP in a novel 'dragging repression mode'. Collectively, these data highlight the dual regulatory mechanisms of PhoP-dependent transcription regulation in governing stress adaptation. These findings provide structural basis for developing potential anti-tuberculosis drugs and/or interventions.


The cryo‐EM structure of E. coli GcvA–TAC. (a) DNA scaffold used in structure determination of E. coli GcvA–TAC. (b, c) Two views of the cryo‐EM density map before post process (b) and structure model (c) of E. coli GcvA–TAC. The EM density maps and cartoon representations of GcvA–TAC are colored as indicated in the color key. NT, non‐template‐strand promoter DNA; T, template‐strand promoter DNA. Orange, GcvAI binding site; cyan, GcvAII binding site; yellow, GcvA activation binding sites (ABS); violet, αCTD binding site; black, −35 element and −10 element.
The interactions between GcvA_DBD and promoter DNA. (a) GcvA_DBD in complex with the gcvB promoter DNA. GcvAI_DBD and GcvAII_DBD are represented as orange or blue cartoon, respectively. (b) Secondary structure of GcvAI_DBD and GcvAII_DBD. (c) Detailed interactions between each GcvA_ DBD protomer and the gcvB promoter DNA. Residues from GcvA_ DBD involved in contacting the upstream or downstream sequences of GcvA binding site are shown in green sticks. (d) Detailed interactions between GcvAI_DBD and GcvAII_DBD. The residues stabilize the dimeric interface between GcvAI_DBD and GcvAII_DBD are shown in orange and blue sticks, respectively. (e) Substitutions of residues included in the GcvA_DBD‐DNA interface and GcvA_DBD dimeric interface reduce in vitro transcription activity. Data for in vitro transcription assays are means of three technical replicates.
The interactions between GcvA_DBD and E. coli RNAP αCTD. (a) Relative locations of E. coli RNAP αCTD, GcvAI_DBD, and the upstream double‐stranded DNA. E. coli RNAP αCTD, GcvAI_DBD are also represented as magenta or orange cartoon, respectively. (b) Structural model of GcvA and E. coli RNAP αCTD. (c) Detailed interactions between GcvAI_DBD and E. coli RNAP αCTD. Hydrogen bonds are shown as orange dashed lines. (d) Mutation of residues involved in the GcvAI_DBD‐αCTD interface suppressed in vitro transcription activity. Data for in vitro transcription assays are means of three technical replicates. Error bars represent ± SEM of n = 3 experiments.
Molecular docking of GcvA_RD with glycine. (A) Molecular docking of GcvA_RD (including RD‐I and RD‐II) with its co‐inducer glycine. GcvA_RD generated from the AlphaFold prediction model (ID: AF‐P0A9F6‐F1) is shown as light blue cartoon. Glycine molecule is shown as spheres. (b) The structural model of GcvA_RD superimposes well on the binary structure of BenM_RD in complex with its inducer molecule (cis‐cis‐muconate) (PDB ID: 2F7A). GcvA_RD and BenM_RD are shown as light blue and pink cartoon. Cis‐cis‐muconate molecule is shown as spheres. (c) Predicted binding pocket for glycine on GcvA_RD. Residues potentially included in interactions with glycine are shown as blue sticks. (d) Mutations of the key residues constituting the predicted inducer binding pocket decreased in vitro transcription activity with the addition of 4 mM glycine. Data for in vitro transcription assays are means of three technical replicates. Error bars represent ± SEM of n = 3 experiments.
Proposed model for LTTR‐dependent transcription activation. (a) Scheme for the observation of the GcvA‐RNAP‐DNA complex via single‐molecule photobleaching assay and two typical trajectories under 3 and 15 mW red laser power separately (upper panel). Dependence of the mean duration of GcvA fluorescence before bleaching on the laser power (lower panel). (b) Two views of the proposed cryo‐EM density map of E. coli GcvA‐TAC assembled by GcvA tetramer. The EM density maps and cartoon representations of GcvA‐TAC are colored as indicated in the color key of Figure 1. (c) Proposed working model for LTTR‐dependent transcription activation. In the absence of co‐inducer, LTTR simultaneously binds RBS and ABS via the DBD dimers, and is unable to activate transcription initiation (upper panel). Upon co‐inducer binding, LTTR tetramerizes, evokes proper conformation changes, and remodels promoter DNA and RNAP, which facilitates formation of a competent transcription activation complex, and efficiently initiate LTTR‐dependent transcription (below panel). Co‐inducer is represented as red balls.
Structural and functional insights into transcription activation of the essential LysR‐type transcriptional regulators

May 2024

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64 Reads

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2 Citations

The enormous LysR‐type transcriptional regulators (LTTRs), which are diversely distributed amongst prokaryotes, play crucial roles in transcription regulation of genes involved in basic metabolic pathways, virulence and stress resistance. However, the precise transcription activation mechanism of these genes by LTTRs remains to be explored. Here, we determine the cryo‐EM structure of a LTTR‐dependent transcription activation complex comprising of Escherichia coli RNA polymerase (RNAP), an essential LTTR protein GcvA and its cognate promoter DNA. Structural analysis shows two N‐terminal DNA binding domains of GcvA (GcvA_DBD) dimerize and engage the GcvA activation binding sites, presenting the −35 element for specific recognition with the conserved σ⁷⁰R4. In particular, the versatile C‐terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σ⁷⁰R4 and promoter DNA, providing more interfaces for stabilizing the complex. Moreover, molecular docking supports glycine as one potential inducer of GcvA, and single molecule photobleaching experiments kinetically visualize the occurrence of tetrameric GcvA‐engaged transcription activation complex as suggested for the other LTTR homologs. Thus, a general model for tetrameric LTTR‐dependent transcription activation is proposed. These findings will provide new structural and functional insights into transcription activation of the essential LTTRs.


Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli

March 2024

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94 Reads

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9 Citations

The ISME Journal

Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.


Fig. 3. Four GlnR molecules engage promoter DNA in M. tuberculosis GlnR-TAC. (A) Relative locations of M. tuberculosis GlnR I _DBD, GlnR II _DBD, GlnR III _DBD, and GlnR IV _DBD located at the upstream double-stranded DNA. (B) Detailed interactions between M. tuberculosis GlnR II _DBD, GlnR I _DBD, and their corresponding GlnR binding sites. The key residues involved are shown as green spheres. (C and D) The relative locations and detailed interactions between GlnR II _DBD and GlnR I _DBD bound to the promoter DNA. The key residues involved in Gln-R II _DBD and GlnR I _DBD are shown as wheat and blue spheres, respectively. (E) Substitutions of GlnR residues involved in promoter engagement reduced in vitro transcription activity. Colors are shown as in Fig. 2.
Fig. 4. The critical protein-protein interactions between M. tuberculosis GlnR and domains of RNAP β flap, σ A R4. (A) Relative locations of M. tuberculosis GlnR I _DBD and RNAP β flap (Left and Middle). GlnR I _DBD and RNAP β flap are shown in surface style (Left). Residues involved between GlnR I _DBD and RNAP β flap are shown as slate (GlnR I _DBD) and cyan spheres (RNAP β flap) (Right). (B) Relative locations of M. tuberculosis GlnR II _DBD and RNAP σ A R4 (Left and Middle). Gln-R II _DBD and RNAP σ A R4 are shown in surface (Left). Residues involved in interactions between GlnR II _DBD and RNAP σ A R4 are shown as yellow (GlnR II _DBD) and wheat spheres (RNAP σ A R4) (Right). RNAP β flap is colored in cyan, and RNAP σ A R4 is colored in yellow. The other colors are shown as in Fig. 2.
Fig. S9). This reveals that the four GlnR-RECs are required for stabilization of GlnR_TAC through synergistically bridging GlnR_DBDs to RNAP.
Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria

May 2023

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61 Reads

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7 Citations

Proceedings of the National Academy of Sciences

In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved β flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.


An SI3-σ arch stabilizes cyanobacteria transcription initiation complex

April 2023

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23 Reads

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12 Citations

Proceedings of the National Academy of Sciences

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σA (σ2) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.


Citations (21)


... The smFRET assay on a TRIF microscope measures the dynamics of the intra-or intermolecular interactions of the surface-immobilized molecules within the 10 nm range (Holden et al., 2010). The smFRET TIRF studies identified the structural dynamics of TFs, Pol II, and nucleosomes during transcription (Andrecka et al., 2008;Chen et al., 2021;Crickard et al., 2017;Kilic et al., 2018;Malkusch et al., 2017;Xiong et al., 2024). Collectively, in vitro single-molecule imaging approaches have provided important kinetic information on the interactions of TFs with chromatin and the assembly of transcription complexes (Table 1). ...

Reference:

Single-molecule imaging for investigating the transcriptional control
Single-molecule reconstruction of eukaryotic factor-dependent transcription termination

... TFs play an important role in inducing downstream functional gene expression and signal transduction [41]. Some cis-acting elements associated with the regulation of plant growth under biotic/abiotic stresses were found in PsAP2/ERFs, such as TC-rich repeats, MBS, MBSI, and LTR ( Fig. 6) (Table S3). ...

Structural and functional insights into transcription activation of the essential LysR‐type transcriptional regulators

... 质粒宿主共进化突变 补偿也是降低适应性代价和提高质粒稳定性的重要方 式. Liu等人 [21] 在共整合质粒的进化实验中发现, 质粒 均显著降低 [120] . 另一方面, 尽管适应性代价可以使耐 药性降低, 但补偿进化和水平转移等降低途径的出现, 使适应性代价逆转耐药性的驱动力变慢甚至停滞 [121] , 从而使耐药性问题持续存在 [122] . ...

Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli
  • Citing Article
  • March 2024

The ISME Journal

... The specific direct repeat units (DRus) PhoP binds are referred as PhoP binding boxes (PHO boxes), each of which carries two conserved PhoP binding sites (site a and site b) separated by several nucleotides (Fig. 1) [34][35][36] . The co-crystal structure of PhoP bound to a PHO box-containing promoter DNA reveals that a PhoP dimer interacts with the PHO box through conserved winged helices, akin to other transcription regulators in the OmpR/PhoB family 12,22,[37][38][39][40] . These interactions facilitate a compact arrangement of the PhoP_RECs and PhoP_DBDs, which restricts a 4-bp spacer between the two binding sites 41 . ...

Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria

Proceedings of the National Academy of Sciences

... The split of RNAP β' subunit also occurs in the PEP core, with the rpoC gene that encodes RNAP β' subunit in E. coli being split in spinach into two genes, rpoC1 and rpoC2, that encode the β' and β'' subunits of PEP, respectively. This split does not result in significant structural differences between the PEP core and bacterial RNAPs ( Supplementary Fig. 4a, b) 28,29 , as the PEP core subunits are similar in sequence to their bacterial RNAP counterparts ( Supplementary Fig. 5) and the structures of the PEP core and bacterial RNAP are also similar (Fig. 2a-c). ...

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex
  • Citing Article
  • April 2023

Proceedings of the National Academy of Sciences

... Consequently, the RNA-DNA base pair at position +5 within the active site is tilted by approximately 20° compared with that in the canonical conformation in other TC structures (Fig. 3b). The tilted RNA-DNA hybrid is reminiscent of that in previously observed paused conformations in mammalian Pol II EC and bacterial RNAP elongation and termination complexes [38][39][40][41] , where the RNA-DNA hybrid adopts a tilted conformation correlated with transcription pausing (Extended Data Fig. 8d). However, no notable accumulation of five-nucleotide products was detected in our in vitro assay ( Fig. 1c and Supplementary Fig. 1a), indicating that this tilted state is not a pausing intermediate. ...

Structural basis for intrinsic transcription termination

Nature

... The vast majority of these predicted sites are non-functional with respect to gene regulation [7,9]. In the intervening years, it has emerged that MarA-like factors often identify DNA targets via an unusual "prerecruitment" mechanism [52][53][54]. Briefly, whilst most transcriptional activators bind their DNA target, and then recruit RNA polymerase, MarA-like proteins can bind RNA polymerase before promoter recognition [55]. ...

Structural basis of three different transcription activation strategies adopted by a single regulator SoxS

Nucleic Acids Research

... Only recently has the structure of P. aeruginosa RNAP in complex with s S and SutA been published. 9 Tools such as aptamers, which specifically recognize P aeruginosa RNAP, can significantly enhance research in several areas, including the purification of RNAP after protein complex reconstitution and the development of biosensors. ...

Pseudomonas aeruginosa SutA wedges RNAP lobe domain open to facilitate promoter DNA unwinding

... Again, when tracking the same distances, we observed that after around 1 μs of simulation time, MarA is again able to bind to the A-box of the mar promoter ( Figure S16). This strongly supports that formation of this disulfide bridge is altering the conformational dynamics of MarA, as well as the structure of the N-terminal HTH motif, in such a way that MarA is no longer able to bind the marbox of the mar promoter (Corbella et al. 2021;Shi et al. 2022), and not simply due to a perturbation effect on the hydrophobic environment. ...

Structural basis of transcription activation by Rob, a pleiotropic AraC/XylS family regulator

Nucleic Acids Research

... Furthermore, it has been shown that the efficiency of the activity of this complex varies depending on the type of miRNAs [126]. Dicer, another key miRNA processing enzyme, cleaves pre-miRNAs into mature double-stranded miRNAs, where one strand is degraded and the other strand binds to the RNA-induced silencing complex (RISC) to fulfill miRNA functions [127]. RBPs can also inhibit the miRNA maturation process. ...

Structural basis of microRNA processing by Dicer-like 1

Nature Plants