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: Activation of bacterial transcription by distortion of promoter base pairing

doi: 10.1093/nar/gkaf1424

Figure Lengend Snippet: MarA activates transcription of flgB from a σ 28 -dependent promoter. ( a ) The σ 70 -dependent promoters flgA P1 and flgB P1 are activated by FlhD 4 C 2 , but not MarA, in vitro . Results of in vitro transcription assays to monitor σ 70 -dependent transcription from flgA P1 and flgB P1. The RNAI transcript is derived from the plasmid replication origin and serves as an internal control. RNA polymerase was used at a concentration of 0.15 μM. Where present, MarA was added at concentrations of 1, 2 , 4, and 5 µM. We used FlhD 4 C 2 at concentrations of 0.05, 0.1, 0.2, and 0.25 µM. In lanes 11–15, FlhD 4 C 2 was used at 0.2 µM concentration. Note that higher transcription factor concentrations can sometimes result in lower overall levels of transcription, indicated by a reduction in RNAI levels. This is likely due to non-specific DNA binding. ( b ) The σ 28 -dependent promoters flgA P2 and flgB P2 are repressed by FlhD 4 C 2 , and the latter activated by MarA, in vitro . As in panel (a) except that σ 28 -associated RNA polymerase was used. Note that this version of RNA polymerase cannot generate the RNAI transcript. ( c ) The σ 28 -dependent flgA P2 promoter is not regulated by MarA in vivo . Results of β-galactosidase assays using T7 express cells carrying pRW50 or derivatives with the indicated flgA::lacZ fusions. Cells also encoded pET21a- fliA to provide low levels of σ 28 due to leaky expression. Cells were grown in LB medium supplemented with salicylic acid (5 µM), to induce MarA expression, or IPTG (1 µM) to induce a short burst of high level of σ 28 production. The results shown are the mean of three independent experiments with error bars showing standard deviation. A two-tailed homoscedastic Student’s t -test was used to calculate P where appropriate; otherwise, a two-tailed test was used (* <.05, ** <.01, and *** <.001). ( d ) The σ 28 -dependent flgB P2 promoter is activated by MarA in vivo . As in panel (c) except that flgB::lacZ fusions were used. P was calculated as in panel (c).

Article Snippet: RNA polymerase core enzyme and RNA polymerase σ 70 holoenzyme were purchased from NEB.

Techniques: In Vitro, Derivative Assay, Plasmid Preparation, Control, Concentration Assay, Binding Assay, In Vivo, Expressing, Standard Deviation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Activation of bacterial transcription by distortion of promoter base pairing

doi: 10.1093/nar/gkaf1424

Figure Lengend Snippet: A defect in opening of the flgB P2 promoter is corrected by MarA-induced base pair distortions. ( a ) Results of a KMnO 4 footprint with different combinations of MarA and σ 28 -associated RNA polymerase. Reactivity to KMnO 4 due to DNA melting at flgA P2 and flgB P2 is shown by open and closed blue triangles, respectively. Equivalent reactivity changes due to MarA are indicated by green arrows. The gel is calibrated with a Maxam–Gilbert sequencing reaction numbered according to the position of the flgB P1 TSS. The TSS locations for flgA P2 and flgB P2 are shown in the adjacent schematic. ( b ) Schematic representation of DNA opening at flgA P2 induced by σ 28 -associated RNA polymerase alone. The schematic shows the DNA strand base sequences for flgA P2 and regions of DNA unwinding indicated in lane 4 of panel (a). The thymine bases reactive to KMnO 4 , in the presence of σ 28 -bound RNA polymerase, are marked by triangles. The promoter −10 element is in blue and the transcription start site (+1) is in upper case and marked by a bent arrow. ( c ) Schematic representation of DNA opening at flgB P2 induced by σ 28 -associated RNA polymerase alone. As in panel (b) except that the flgB P2 sequence and opening pattern (in the absence of MarA) are shown. Note that a portion of the MarA site (green) is also highlighted. (d) Schematic representation of DNA opening at flgB P2 induced by MarA and σ 28 -associated RNA polymerase. As in panel (b) except that data are shown for reactions with MarA. The position of KMnO 4 reactivity induced by MarA is highlighted by a green triangle.

Article Snippet: RNA polymerase core enzyme and RNA polymerase σ 70 holoenzyme were purchased from NEB.

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Activation of bacterial transcription by distortion of promoter base pairing

doi: 10.1093/nar/gkaf1424

Figure Lengend Snippet: Artificial disruption of flgB P2 promoter base pairing removes the requirement for MarA. ( a ) Results of a KMnO 4 footprint with σ 28 -associated RNA polymerase and variants of flgB P2. Reactivity to KMnO 4 , due to DNA melting at flgB P2, is shown by closed blue triangles. The flgB P2 m variant (lanes 6–8) has a single base mismatch that prevents base pairing at the location indicated by the red triangle. This is the same base pair that MarA protein perturbs upon binding the DNA. The gel is calibrated with a sequencing reaction and numbering is with respect to the flgB P1 TSS. ( b ) Schematic representation of the wild type flgB P2 sequence. The top and bottom panels indicate the degree of DNA unwinding in the presence and absence of σ 28 -bound RNA polymerase [equivalent to panel (a) lanes 3 and 4]. Triangles indicate that the thymine bases reactive to KMnO 4 in the presence of RNA polymerase bound with σ 28 . (c) Schematic representation of an flgB P2 m variant having single base pair mismatch to facilitate pre-opening of the DNA. The top and bottom panels indicate the degree of DNA unwinding in the presence and absence of σ 28 -bound RNA polymerase [equivalent to panel (a) lanes 7 and 8]. The altered nucleotide is shown in red. Triangles indicate that the thymine bases reactive to KMnO 4 in the presence of RNA polymerase bound with σ 28 .

Article Snippet: RNA polymerase core enzyme and RNA polymerase σ 70 holoenzyme were purchased from NEB.

Techniques: Disruption, Variant Assay, Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Activation of bacterial transcription by distortion of promoter base pairing

doi: 10.1093/nar/gkaf1424

Figure Lengend Snippet: Structural modelling of the ternary complex formed between flgB P2, MarA, and σ 28 -associated RNA polymerase. ( a ) Structural model of MarA and the σ 28 RNA polymerase holoenzyme in the context of the flgBP2 open complex. The σ 28 factor is coloured blue, and labelled alongside other RNA polymerase subunits, while MarA is shown as green. Where needed, proteins are partially transparent to allow DNA (grey) trajectory to be visualized. The promoter −10 and −35 elements recognized by σ 28 are in blue on the top strand and base pairs distorted upon MarA binding are highlighted red. ( b ) An expansion with only MarA and σ 28 visible. Promoter elements are labelled. Note that the −10 element base pair, at position −11, forms the upstream boundary of DNA opening and is targeted for distortion by MarA.

Article Snippet: RNA polymerase core enzyme and RNA polymerase σ 70 holoenzyme were purchased from NEB.

Techniques: Binding Assay