e coli rnap core enzyme  (New England Biolabs)

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: bioRxiv

Article Title: Structural basis for intrinsic transcription termination

doi: 10.1101/2022.08.30.505822

Figure Lengend Snippet: (A) The sequences of the nucleic-acid scaffold used for cryo-EM study. (B) The cryo-EM map (left) and structural model (right) for TTC-pause complex. The RNAP subunits and nucleic-acid chains are colored as in the color scheme. (C) The cryo-EM map for the nucleic-acid scaffold. (D) The comparison of the RNA–DNA translocation states among TTC-pause complex, half-translocation state in a hairpin-stabilized paused transcription elongation complex ( his PEC; PDB: 6ASX), a pre-translocation complex (PDB: 4YLN), and a post-translocation complex (PDB: 6XLN). (E) The consensus sequence of bacterial intrinsic terminators (upper panel) and the schematic presentation of the nucleotides of the transcription bubble in the TTC-pause complex. (F) The conformational comparison among TTC-pause complex, TEC (PDB: 6ALF), and his PEC (PDB: 6ASX). (G) The cryo-EM map and structural model of ntDNA of the transcription bubble. The insert shows the interaction between the flipped A (–4) nucleotide and the ‘NT–4’ pocket.

Article Snippet: E. coli RNAP core enzyme for cryo-EM and most in vitro assays was over-expressed from E. coli BL21(DE3) (Novo protein, Inc.) carrying p Ec ABC and pCDF- Ec rpoZ (Table S1) and purified as described .

Techniques: Cryo-EM Sample Prep, Comparison, Translocation Assay, Sequencing

Journal: bioRxiv

Article Title: Structural basis for intrinsic transcription termination

doi: 10.1101/2022.08.30.505822

Figure Lengend Snippet: (A) The nucleic-acid scaffold and the antisense RNA (asRNA) used for cryo-EM structure determination. (B) The cryo-EM map (left) and structural model (right) for TTC-hairpin complex. (C) The cryo-EM map for nucleic-acid scaffold. The left insert shows the map for the RNA duplex in the RNA exit channel and the right insert shows the superimposition of the base pairs (–3 to 1) of the RNA–DNA hybrid between TTC-hairpin (colored as in the scheme) and TTC-pause (gray). (D) RNA duplex in the RNA exit channel. The electrostatic potential surface of RNAP was generated using APBS tools in Pymol. (E) The detailed interaction of the RNA duplex with residues in the RNA exit channel. Spheres, the Cβ atom of polar residues in H-bond distance with the phosphate backbone of RNA duplex. (F) The interaction of the –11 base pair of RNA duplex with residues in the RNA exit channel. (G) Further extension of RNA duplex is blocked by RNAP β′ ZBD, β′ lid, and β Sw3 motifs. (H) The structural comparison of RNA exit channel between TTC-pause (gray) and TTC-hairpin (colored). (I) The global conformational movement of TTC-hairpin (colored) compared with TTC-pause (gray). (J) The comparison of the first three base pairs of the RNA–DNA hybrid between TTC-hairpin (pink and blue) and TEC (gray) (PDB: 6ALF). The two structures were superimposed based in the RNAP-β′ lid and rudder motifs. (K) The G (–10) of the tDNA is under the tunnel formed by RNAP-β′ lid and rudder and ready to pair with the –10 nucleotide of ntDNA.

Article Snippet: E. coli RNAP core enzyme for cryo-EM and most in vitro assays was over-expressed from E. coli BL21(DE3) (Novo protein, Inc.) carrying p Ec ABC and pCDF- Ec rpoZ (Table S1) and purified as described .

Techniques: Cryo-EM Sample Prep, Generated, Comparison

Journal: bioRxiv

Article Title: Structural basis for intrinsic transcription termination

doi: 10.1101/2022.08.30.505822

Figure Lengend Snippet: (A) The asRNA-induced termination strategy used for obtaining the cryo-EM structure of TTC-release. (B) The results of RNA release assay show that asRNA12 induces release of nascent RNA in a time-dependent manner. T, total fraction; S, supernatant fraction. (C) The cryo-EM map and structural model of TTC-release. (D) The cryo-EM map and model of the nucleic-acid scaffold TTC-release. (E) The comparison of transcription bubble sizes of TTC-release and TTC-hairpin. (F) The rewound 5-bp dsDNA (–10 to –6) were loosely restrained by β protrusion, β′ clamp helices, and β′ rudder and further rewinding of the upstream dsDNA is stopped by the β lobe domain due to a closed clamp. (G) The in vitro transcription assay suggests that DNA unwinding is required for intrinsic termination at the λ tR2 terminator. Data are presented as mean ± SEM, n=3 biologically independent experiments. **P<0.01, ***P<0.001, ****P<0.0001. (H) RNA releases from TTC during termination faster than DNA releases. A TTC scaffold making all RNAP contacts with P label on the T strand 5′ O and RNA 3′ phosphodiester was immobilized on beads. Upon asRNA addition, the rate of RNA release was 0.037 ± 0.006 s −1 whereas DNA released at 0.0075 ±0.001 s −1 .

Article Snippet: E. coli RNAP core enzyme for cryo-EM and most in vitro assays was over-expressed from E. coli BL21(DE3) (Novo protein, Inc.) carrying p Ec ABC and pCDF- Ec rpoZ (Table S1) and purified as described .

Techniques: Cryo-EM Sample Prep, Release Assay, Comparison, In Vitro, Transcription Assay