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- PDB-8y6u: Cryo-EM structure of E.coli transcription initiation complex with... -

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Basic information

Entry
Database: PDB / ID: 8y6u
TitleCryo-EM structure of E.coli transcription initiation complex with transcription factor GcvA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Glycine cleavage system transcriptional activator
  • Non-template promoter DNA
  • RNA polymerase sigma factor RpoD
  • Template promoter DNA
KeywordsTRANSCRIPTION/DNA / RNA polymerase / TRANSCRIPTION-DNA COMPLEX
Function / homology
Function and homology information


RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / response to UV ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / response to UV / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / sequence-specific DNA binding / intracellular iron ion homeostasis / protein dimerization activity / DNA-binding transcription factor activity / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / metal ion binding / cytoplasm / cytosol
Similarity search - Function
LysR, substrate-binding / LysR substrate binding domain / LysR-type HTH domain profile. / Transcription regulator HTH, LysR / Bacterial regulatory helix-turn-helix protein, lysR family / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 ...LysR, substrate-binding / LysR substrate binding domain / LysR-type HTH domain profile. / Transcription regulator HTH, LysR / Bacterial regulatory helix-turn-helix protein, lysR family / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Glycine cleavage system transcriptional activator / RNA polymerase sigma factor RpoD
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsLin, W. / Shi, J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81903526 China
Citation
Journal: Biochem Biophys Res Commun / Year: 2024
Title: NMR analysis of a loop-bulge structure of UUCGA pentaloop.
Authors: Rika Ichijo / Gota Kawai /
Abstract: Although structures of many RNA loops, such as GNRA and UNCG tetraloops, were well known, it is still possible to find more RNA structures. In the present study, solution structure of an RNA fragment ...Although structures of many RNA loops, such as GNRA and UNCG tetraloops, were well known, it is still possible to find more RNA structures. In the present study, solution structure of an RNA fragment having UUCGA pentaloop was analyzed by NMR spectroscopy. It was found that the UUCG tetraloop is formed and the adenosine residue at the 3' side of the tetraloop is bulged out. The characteristic motif of the loop-bulge structure has also been found in other RNAs including CUUGU and CUGGC pentaloops. Along with the recently found T-hairpin structure with a UUUGAUU loop, in which UUUGA pentaloop and UU bulge are formed, the loop-bulge structures can be categorized as an RNA motif and it may be called as the integrated structure loop, I-loop.
#1: Journal: Protein Sci / Year: 2024
Title: Structural and functional insights into transcription activation of the essential LysR-type transcriptional regulators.
Authors: Jing Shi / Zhenzhen Feng / Qian Song / Fulin Wang / Zhipeng Zhang / Jian Liu / Fangfang Li / Aijia Wen / Tianyu Liu / Zonghang Ye / Chao Zhang / Kalyan Das / Shuang Wang / Yu Feng / Wei Lin /
Abstract: 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 ...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 σR4. In particular, the versatile C-terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σR4 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.
History
DepositionFeb 3, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Glycine cleavage system transcriptional activator
J: Glycine cleavage system transcriptional activator
1: Non-template promoter DNA
2: Template promoter DNA
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor RpoD
I: DNA-directed RNA polymerase subunit alpha


Theoretical massNumber of molelcules
Total (without water)622,25911
Polymers622,25911
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 3 molecules HJF

#1: Protein Glycine cleavage system transcriptional activator / Gcv operon activator


Mass: 34447.500 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: gcvA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A9F6
#8: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 70352.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoD, GHR40_08205, NCTC12650_00972 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q0P6L9

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DNA chain , 2 types, 2 molecules 12

#2: DNA chain Non-template promoter DNA


Mass: 28330.164 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain Template promoter DNA


Mass: 28584.369 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)

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DNA-directed RNA polymerase subunit ... , 4 types, 6 molecules ABICDE

#4: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150804.922 Da / Num. of mol.: 1 / Mutation: D516V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E.coli transcription initiation complex with GcvA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 583044 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00334661
ELECTRON MICROSCOPYf_angle_d0.54147386
ELECTRON MICROSCOPYf_dihedral_angle_d18.0185692
ELECTRON MICROSCOPYf_chiral_restr0.0395410
ELECTRON MICROSCOPYf_plane_restr0.0035699

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