Journal: bioRxiv

Article Title: Distinct spatial organisation of Rho and RNA Polymerase in Salmonella cells

doi: 10.64898/2026.05.02.722398

Figure Lengend Snippet: Bacteria were grown to an OD600 of 0.2 (exponential phase) or to an OD600 of 2 (stationary phase), fixed with paraformaldehyde, and processed for structured illumination microscopy (SIM) as described in Experimental Procedures. Images shown are representative of the observed localisation patterns across multiple cells and independent experiments. A,B. Strain MA12189 ( rho :: sfGFP rpoC - mCherry ) in exponential phase ( A ) and stationary phase ( B ). C,D . Strain MA14978 ( rho Δ48:: mCherry rpoC - sfGFP ) in exponential phase ( C ) and stationary phase ( D ). In exponential phase, Rho and RNAP (RpoC) show patchy fluorescence patterns with only partial spatial overlap. In stationary phase, RNAP patches become more diffuse and remain associated with a compacted nucleoid, whereas Rho accumulates toward the cell periphery. Both reciprocal labelling arrangements yield similar patterns, suggesting that Rho’s spatial organisation is independent of the fluorophore used

Article Snippet: E. coli RNA polymerase (RNAP) holoenzyme was purchased from New England Biolabs.

Techniques: Bacteria, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) 2D electrophoresis ladder used to annotate the cellular-extracted T7A1U plasmid. ( B-C ) 2D electrophoresis gel of cellular-extracted T7A1U plasmid and the same plasmid with RNAP and TopI after 30 minutes of reaction, respectively. ( D ) Gel quantification of the dominant ΔLk number in B (top) and C (bottom). ( E ) Map resolution evaluation for a representative particle in . Resolution was estimated by measuring the Fourier shell correlation (FSC) between two independently determined half-maps (even-odd dose frames, pink) at 0.143 and between the map and fitted model (blue) at 0.5. ( F ) Model fitting workflow, see also materials and methods. ( G ) Three rotational views of a pUC19-T7A1U plasmid particle showing the variation in the number of DNA crossings. The DNA crossing positions are indicated by tan-colored arrowheads.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Two-Dimensional Gel Electrophoresis, Plasmid Preparation

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) Cryo-ET map and model of the –sc. plasmids bound with stalled RNAPs under PS condition. The top panel shows an exemplary particle (left) alongside zoomed-in views of the modeled RNAP stalling sites (right). The bottom panel features a collection of stalled TECs, with RNAP highlighted by purple arrowheads. ( B ) The superimposition of bound RNAPs reveal apical DNA dynamics. A dashed vector along the fissure composed of β′, α II (purple) and β, α I (cyan) subunits was used to depicted RNAP orientation. ( C )The superimposition of the apical DNA segments shows the RNAP orientation dynamics (purple arrow heads). ( D ) Schematic highlighting different DNA geometries: TEC on –sc. DNA template (left), and TIC (middle) and TEC (right) on linear DNA templates. The dashed-orange line represents the flexible portion of the DNA. ( E ) An electrophoresis gel shows reduced abortive transcript levels on –sc. DNA compared to linear DNA toward the stalling site. ( F ) Control for A, with RNAP replaced by dCas9 (orange). ( G and H ) Superimposition of bound dCas9 and apical DNA, respectively, from all reconstructed particles. ( I ) Plasmid apex number distribution. ( J ) Schematic shows dCas9’s smaller pocket curvature compared to that of RNAP, reshaping bound plasmids with enlarged distal ends and tightly intertwined body.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Tomography, Plasmid Preparation, Electrophoresis, Control

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) Two orthogonal views of the sub-tomogram averaged RNAP map fitted with PDB structure 6ALH. The total number of RNAPs (n =1781) selected from the tomogram yielded an averaged map resolution of 14.1 Å (top panel), while using only the plasmid-bound RNAP population (n =232) result in a resolution of 17.7 Å (bottom panel). ( B ) Superimposition of TIC DNA (4YLN, 6CA0, 6JBQ, and 6N60, labeled with gold color) with TEC DNA (6ALH, labeled with red color). Both complexes utilize a short linear DNA template. ( C ) Superimposition of the consensus DNA trajectory of the apically stalled TEC with –sc. DNA template onto B. The downstream and upstream DNA were color in green and yellow, respectively. ( D ) Sub-tomogram averaged dCas9 map fitted with PDB structure 6O0X. A total of 875 dCas9 molecules selected from the tomogram yielded an average map resolution of 17.6 Å (top panel), while the plasmid-bound subset (n = 116) achieved 18.9 Å (bottom panel). Resolutions were estimated by measuring the Fourier shell correlation (FSC) between two independently determined half-maps at 0.143.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Plasmid Preparation, Labeling

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A-D ) Statistical analysis of pUC19-T7A1U plasmid (P.) cylindricity, radius of gyration, sphericity, and writhe number with binding enzymes, respectively, in the presence of stalled RNAP (RNAP S.) or dCas9. ( E ) Tracing of plasmid curvature near apices with traces aligned to the apices (index zero). ( F ) Statistical comparison of plasmid apical and intermediate region curvature distribution. ( G ) Traces of the plectoneme width (DNA spacing) starting from each apex (index zero). ( H ) Statistical comparison of the plectoneme width distribution. ( I ) Overlay of apical bound RNAPs (top) and labeling their RNA protrusion directions in pink (bottom). ( J-M ) Statistical analyses mirror those in ( A-D ) for plasmids with transcribed RNAP (RNAP T.). ( N ) Schematics showing the segmentation of plectoneme branches (left four panels) followed by branch Writhe density calculation (right panel): (i) DNA colorization based apices, (ii) plectoneme central axis determination, (iii) branch segmentation and contour length calculation, and (iv) branch Writhe number calculation. The writhe density was calculated by dividing the writhe number of each branch segmentation by the contour length of the segment. ( O ) Plectoneme contour length distribution for TECs. Statistics are calculated using a Mann-Whitney test, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; and ns, not significant.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Plasmid Preparation, Binding Assay, Comparison, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) Cryo-ET 3D map and model of –sc. plasmid in the presence of RNAP (purple) after 10 min of transcription under PS conditions. ( B ) Zoom-in image of two representative apically bound RNAPs, with nascent RNA protrusion densities highlighted by black arrows. ( C ) The superimposition of apical DNA segments. ( D ) Z-dimensional slice (10 nm thickness) of sub-tomograms showing plasmid bound RNAPs after (top) and before (bottom) adding full set of NTPs. Each slice are superimposed with its fitted plasmid model. RNA density is marked by red dashed circles. ( E ) Plasmid apex number distribution. ( F-G ) Electrophoresis analysis of RNAP transcription on nicked and –sc. templates over time, with quantification of their pause release dynamics, respectively. ( H ) Schematic illustration of transcription-induced new plectoneme formation on –sc. plasmid. RNAPs are represented as purple pentagons. DNA translational and rotational motion is indicated by green arrows, with orange color denoting drags. Plus and minus signs indicate positive and negative DNA torsion, respectively.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Tomography, Plasmid Preparation, Electrophoresis

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) Cryo-ET 3D maps and models showing –sc. plasmids simultaneously bound with stalled RNAP (purple) and dCas9 (orange) under PS condition. Representative unbranched and branched particles displayed in the top panel, with their respective particle collections presented in the bottom panel. ( B ) RNAP release from stalling in A observed after 10 minutes of transcription. ( C ) Comparison of off-apex particle ratios for RNAP and dCas9. ( D ) Schematic displaying three apical configurations of dCas9 (left) and illustrating the accumulation and release of torsion in a –sc. plasmid due to simultaneous bindings of dCas9 (orange triangles) and RNAP (purple pentagons) on apices (right). ( E ) Quantification of multi-RNAP and dCas9 binding plasmids ratio within the population. ( F ) Schematics depicting the active RNAP transcription induced topological domains and the supercoiling rearrangement in the presence of torsional block of dCas9. ( G ) Quantification of plasmids’ apex number. ( H ) Assessment of RNAP pause release in the presence of dCas9 during transcription on nicked and –sc. templates over time via electrophoresis. The RNA band intensities were quantified at the stalling site and three strong pausing sites.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Tomography, Comparison, Plasmid Preparation, Binding Assay, Blocking Assay, Electrophoresis

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: (A) Two orthogonal views of the superimposed apical DNA segments of the plasmid, illustrating the dynamics of stalled-RNAP (purple vectors, left panel) and dCas9 (orange vectors, right panel) in their co-presence on the plasmid. (B) Two orthogonal views of the superimposed apical DNA segments of the plasmid, illustrating the dynamics of transcribed-RNAP (purple vectors) and dCas9 (orange vectors) in their co-presence on the plasmid. ( C-F ) Statistical analysis of plasmid (P.) cylindricity, radius of gyration, sphericity, and writhe number with binding enzymes, respectively, in the presence of transcribed/stalled RNAP (RT./RS.) and dCas9 (Cas). Statistics are calculated using a Mann-Whitney test, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; and ns, not significant. ( G ) Tracing of plasmid curvature near apices with traces aligned to the apices (index zero). ( H ) Statistical comparison of plasmid apical and intermediate region curvature distribution. ( I ) Traces of the plectoneme width (DNA spacing) starting from each apex (index zero). ( J ) Statistical comparison of the plectoneme width distribution. ( K ) Quantification of writhe density in plasmid branches. ( L ) Mapping all bound RNAPs on pUC19-T7A1U templates using dCas9 as a fiducial. RNAP on the transcriptional region, oriented downstream, is marked in pink; RNAP on the non-transcriptional region or pointing upstream, is colored in tan and blue, respectively.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Plasmid Preparation, Binding Assay, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A-C ) Electrophoresis assay of RNAP transcription on –sc. and nicked templates in the presence of dCas9 and on –sc. template in the presence of TopI over time, respectively. ( D-G ) Statistical analysis of plasmid (P.) cylindricity, radius of gyration, sphericity, and writhe number with binding enzymes, respectively, in the presence of transcribed RNAP (RT.) and Topoisomerase I (Top). ( H ) Tracing of plasmid curvature near apices with traces aligned to the apices (index zero). ( I ) Statistical comparison of plasmid apical and intermediate region curvature distribution. ( J ) Traces of the plectoneme width (DNA spacing) starting from each apex (index zero). ( K ) Statistical comparison of the plectoneme width distribution. Statistics are calculated using a Mann-Whitney test, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; and ns, not significant.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Electrophoresis, Plasmid Preparation, Binding Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: ( A ) Schematic representation of TopI binding to RNAP β′ subunit or DNA. ( B ) Assessment of RNAP pause release in the presence of TopI via electrophoresis. ( C ) Cryo-ET image of a z-dimensional slab (50 nm thickness) of the sample after 10 minutes of transcription of the pUC19-T7A1 in the presence of TopI. ( D ) Cryo-ET 3D reconstructions of the above plasmid in the presence of RNAP and TopI after 10 min of transcription. The left and right panels display unbranched vs. branched particles, respectively. Apical and non-apical binding RNAP are distinguished with purple and green arrowheads, respectively. ( E ) Zoom-in image of two representative plasmid-bound RNAPs that escape from the apical location of the plasmid. ( F-G ) Quantification of the plasmid’s apex number and non-apical RNAP ratio, respectively. ( H ) The superimposition of plasmid-bound RNAPs (top panel) and RNAP’s upstream and downstream DNA segments (bottom panel). ( I-J ) DNA supercoiling relaxation assay in the presence of RNAP and TopI at ratios of 3:1 and 1:3, respectively. ( K-L ) Quantification of DNA Linking number in I and J, respectively.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Binding Assay, Electrophoresis, Tomography, Plasmid Preparation

Journal: bioRxiv

Article Title: Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

doi: 10.64898/2026.03.25.714350

Figure Lengend Snippet: Schematic diagram illustrating the transcription cycle modulated by supercoiling and apical binding: (I) Enhancement of –sc. in the plasmid by enzymes like DNA gyrase imposes apical constraints on RNAP, facilitating transcription initiation. (II) Apically initiated RNAP facilitates spatial RNA-DNA separation, while apically bound regulatory proteins promote topological domains during transcription. (III) Hyper-negative supercoiling domain prompts multi-RNAP loading. Removal of torsional blocks prevents unbound torsional buildup during transcription. (IV) Activation of enzyme, such as TopI, partially releases negative torsion in the plasmid, sufficient to dislodge RNAP from apices and facilitate transcription elongation. The green and blue arrows indicate upregulation and downregulation, respectively.

Article Snippet: Stalled elongation complexes labeled with radiolabeled nascent RNA were formed by assembling E.coli RNA polymerase Holoenzyme (10 pmol, New England Biolabs) in pUC19-T7A1-U (1 pmol) TB40 buffer (20 mM Tris pH 8.0, 40 mM KCl, 5 mM MgCl 2 , 0.02 mg/mL BSA, and 1 mM DTT) in the presence of 10 μM of ATP, GTP, CTP (Thermo Scientific, R0481) and 50 μM of GpA dinucleotide (TriLink Biotechnologies) and α-P32-ATP (Perkin Elmer).

Techniques: Binding Assay, Plasmid Preparation, Activation Assay

Journal: Nucleic Acids Research

Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

doi: 10.1093/nar/gkag102

Figure Lengend Snippet: Development and validation of NADbio-northern blotting analysis. ( A ) sibD gene produces NAD-RNAs. The NAD cap content in various RNA samples was determined with LC–MS. The samples analyzed included purified sibD transcripts from the E. coli K12 strain, along with 10 nM NAD + standard. One hundred nanograms of ppp-SibD RNA and NAD-SibD RNA from IVT products were used as negative and positive controls, respectively. ( B ) A schematic illustration of the workflow for the NADbio-northern blotting analysis. NAD and biotin are highlighted in red and blue, respectively. ‘DIG’ highlighted in yellow indicates the digoxin tag in the DNA probes. ( C ) Detection of NAD-RNAs by NADbio-northern blotting with synthetic 5′-ppp-SibD and NAD-SibD. HRP-streptavidin blotting was performed to monitor biotinylation of RNAs mediated by the ADPRC-SPAAC reaction. In addition, a digoxin-tagged sibD DNA probe was used to detect sibD transcripts. ‘ADPRC+’ indicates the biotinylation of NAD-RNAs via the ADPRC-SPAAC reaction with ADPRC, while ‘ADPRC-’ denotes the ADPRC-SPAAC reaction without ADPRC. ( D ) Detection of several NAD-capped cellular RNAs with NADbio-northern blotting analysis. Total RNAs isolated from the wild-type strain were subjected to NADbio-northern blotting analysis. Individual RNAs in the eluate were detected with digoxin-conjugated gene-specific probes. The bands corresponding to individual NAD-RNAs in ADPRC+ lanes were marked with red arrows.

Article Snippet: To perform IVT assays with various sigma factors, a similar assay was conducted, except the E. coli RNAP holoenzyme was replaced by the same amount of E. coli RNAP core enzyme (NEB) and in the absence of ppGpp or DksA.

Techniques: Biomarker Discovery, Northern Blot, Liquid Chromatography with Mass Spectroscopy, Purification, Isolation

Journal: Nucleic Acids Research

Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

doi: 10.1093/nar/gkag102

Figure Lengend Snippet: Several RNAs transcribed by the sibD minimal promoter from E. coli chromosomal DNA could be NAD capped. ( A ) Schematic illustration of gene editing designs in the E. coli genome for four small RNAs expression driven by the sibD minimal promoter. The gene body of trpT is highlighted in purple, sroC in green, and ryjA and symR in blue. The sibD minimal promoter ( sibD P-35 ) is labelled as a short red line, and the rrnB terminator is highlighted in yellow. ( B ) Detection of NAD caps in SibD, TrpT, RyjA, SroC, and SymR RNAs with NADbio-northern blotting in the wild-type and indicated mutant strains. ‘ADPRC+’ indicates the biotinylation of NAD-RNAs via the ADPRC-SPAAC reaction with sufficient ADPRC, while ‘ADPRC−’ denotes the ADPRC-SPAAC reaction without ADPRC. 5S RNAs were detected as loading controls. ( C ) Detection and quantification of NAD-RNAs from SibD, TrpT, RyjA, SroC, and SymR using APB gel blotting. Capping ratios were calculated based on the band intensity of the capped transcripts relative to the total transcripts (both capped and uncapped transcripts) in the APB gel.

Article Snippet: To perform IVT assays with various sigma factors, a similar assay was conducted, except the E. coli RNAP holoenzyme was replaced by the same amount of E. coli RNAP core enzyme (NEB) and in the absence of ppGpp or DksA.

Techniques: Expressing, Northern Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

doi: 10.1093/nar/gkag102

Figure Lengend Snippet: Effects of (p)ppGpp and DksA on transcription and NAD capping of certain small RNAs in E. coli cells. ( A ) The NAD capping level of SibD increased upon transient induction of RelA 455aa and DksA. Both RelA 455aa and DksA were expressed from plasmids under the control of the pBAD promoter. NAD capping of SibD was assessed by APB gel blotting. The total level of SibD RNA in each lane was quantified from the normal gel using ImageJ software and normalized to the intensity in the first EV lane. The NAD capping ratio was calculated as the percentage of the intensity of the NAD-capped band relative to the sum of the intensities of both the capped and uncapped bands in the APB gel. ‘Arabinose−’ indicates RNA samples without arabinose induction, while ‘Arabinose+’ signifies that arabinose was added to induce the expression of RelA 455aa and DksA. ‘EV’ indicates strain carrying the empty pBAD33.1 vector. The tmRNA was used as a loading control and each blotting has three independent replicates. ( B ) Detection of NAD-capped transcripts of five sRNAs with NADbio-northern blotting analysis, including four known NAD-RNAs: SibC, SibD, SibE, and GcvB. The tmRNA was used as a loading control. ( C – G ) Detection of total transcripts and NAD-capped transcripts of five sRNAs, namely SibA, SibC, SibD, SibE, and GcvB, respectively. The total abundance of individual RNA was determined by electrophoresis on a standard PAGE gel followed by northern blotting (labelled as normal gel), while the NAD-capped transcripts were identified with APB gel blotting (labelled as APB gel). The non-NAD-RNA SibA was included as a negative control. The NAD capping ratio was calculated based on the band intensity of the NAD-capped version relative to the total transcription levels (NAD-capped version plus uncapped version). Two types of synthetic RNAs for each sRNA, namely with 5′-ppp- and 5′-NAD modifications, were used as controls.

Article Snippet: To perform IVT assays with various sigma factors, a similar assay was conducted, except the E. coli RNAP holoenzyme was replaced by the same amount of E. coli RNAP core enzyme (NEB) and in the absence of ppGpp or DksA.

Techniques: Control, Software, Expressing, Plasmid Preparation, Northern Blot, Electrophoresis, Negative Control

Journal: Nucleic Acids Research

Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

doi: 10.1093/nar/gkag102

Figure Lengend Snippet: Several RNAs transcribed by the sibD minimal promoter from E. coli chromosomal DNA could be NAD capped. ( A ) Schematic illustration of gene editing designs in the E. coli genome for four small RNAs expression driven by the sibD minimal promoter. The gene body of trpT is highlighted in purple, sroC in green, and ryjA and symR in blue. The sibD minimal promoter ( sibD P-35 ) is labelled as a short red line, and the rrnB terminator is highlighted in yellow. ( B ) Detection of NAD caps in SibD, TrpT, RyjA, SroC, and SymR RNAs with NADbio-northern blotting in the wild-type and indicated mutant strains. ‘ADPRC+’ indicates the biotinylation of NAD-RNAs via the ADPRC-SPAAC reaction with sufficient ADPRC, while ‘ADPRC−’ denotes the ADPRC-SPAAC reaction without ADPRC. 5S RNAs were detected as loading controls. ( C ) Detection and quantification of NAD-RNAs from SibD, TrpT, RyjA, SroC, and SymR using APB gel blotting. Capping ratios were calculated based on the band intensity of the capped transcripts relative to the total transcripts (both capped and uncapped transcripts) in the APB gel.

Article Snippet: To perform IVT assays with various sigma factors, a similar assay was conducted, except the E. coli RNAP holoenzyme was replaced by the same amount of E. coli RNAP core enzyme (NEB) and in the absence of ppGpp or DksA.

Techniques: Expressing, Northern Blot, Mutagenesis

Journal: Nucleic Acids Research

Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp

doi: 10.1093/nar/gkag102

Figure Lengend Snippet: Effects of (p)ppGpp and DksA on transcription and NAD capping of certain small RNAs in E. coli cells. ( A ) The NAD capping level of SibD increased upon transient induction of RelA 455aa and DksA. Both RelA 455aa and DksA were expressed from plasmids under the control of the pBAD promoter. NAD capping of SibD was assessed by APB gel blotting. The total level of SibD RNA in each lane was quantified from the normal gel using ImageJ software and normalized to the intensity in the first EV lane. The NAD capping ratio was calculated as the percentage of the intensity of the NAD-capped band relative to the sum of the intensities of both the capped and uncapped bands in the APB gel. ‘Arabinose−’ indicates RNA samples without arabinose induction, while ‘Arabinose+’ signifies that arabinose was added to induce the expression of RelA 455aa and DksA. ‘EV’ indicates strain carrying the empty pBAD33.1 vector. The tmRNA was used as a loading control and each blotting has three independent replicates. ( B ) Detection of NAD-capped transcripts of five sRNAs with NADbio-northern blotting analysis, including four known NAD-RNAs: SibC, SibD, SibE, and GcvB. The tmRNA was used as a loading control. ( C – G ) Detection of total transcripts and NAD-capped transcripts of five sRNAs, namely SibA, SibC, SibD, SibE, and GcvB, respectively. The total abundance of individual RNA was determined by electrophoresis on a standard PAGE gel followed by northern blotting (labelled as normal gel), while the NAD-capped transcripts were identified with APB gel blotting (labelled as APB gel). The non-NAD-RNA SibA was included as a negative control. The NAD capping ratio was calculated based on the band intensity of the NAD-capped version relative to the total transcription levels (NAD-capped version plus uncapped version). Two types of synthetic RNAs for each sRNA, namely with 5′-ppp- and 5′-NAD modifications, were used as controls.

Article Snippet: To perform IVT assays with various sigma factors, a similar assay was conducted, except the E. coli RNAP holoenzyme was replaced by the same amount of E. coli RNAP core enzyme (NEB) and in the absence of ppGpp or DksA.

Techniques: Control, Software, Expressing, Plasmid Preparation, Northern Blot, Electrophoresis, Negative Control