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- PDB-9zp2: E. coli RNA polymerase elongation complex containing the unnatura... -

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Basic information

Entry
Database: PDB / ID: 9zp2
TitleE. coli RNA polymerase elongation complex containing the unnatural dP:Z*TP base pair in a trigger-loop-open conformation (b)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template strand DNA
  • RNA nonamer
  • Template strand DNA
KeywordsTRANSCRIPTION/DNA/RNA / E. coli RNA polymerase elongation complex containing the unnatural dP:Z*TP base pair in a trigger-loop-open conformation (b) / TRANSCRIPTION / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / nitrate assimilation / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / bacterial-type flagellum-dependent cell motility / DNA-directed RNA polymerase complex ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / nitrate assimilation / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / bacterial-type flagellum-dependent cell motility / DNA-directed RNA polymerase complex / cell motility / DNA-templated transcription elongation / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA-templated transcription / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime ...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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / 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 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / 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 polymerase Rpb1, domain 1 / RNA polymerases D / 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, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / 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
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsLi, Q. / Benner, S.A. / Wang, D.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM102362 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM148476 United States
National Science Foundation (NSF, United States)MCB-2419300 United States
National Science Foundation (NSF, United States)MCB-1939086 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R24GM154186 United States
CitationJournal: Nat Commun / Year: 2026
Title: Structural basis of transcription of the hachimoji eight-letter alphabet by E. coli RNA polymerase.
Authors: Qingrong Li / Hyo-Joong Kim / Yan Liu / Juntaek Oh / Peini Hou / Shuichi Hoshika / Grigore Pintilie / Sriram Aiyer / Jenny Chong / Dmitry Lyumkis / Steven A Benner / Dong Wang /
Abstract: Expanded genetic alphabets with synthetic nucleotides can greatly increase the chemical diversity of nucleic acids, enabling new molecular functions. Because cellular transcription is executed by ...Expanded genetic alphabets with synthetic nucleotides can greatly increase the chemical diversity of nucleic acids, enabling new molecular functions. Because cellular transcription is executed by multi-subunit RNA polymerases, the compatibility of unnatural base pairs with this machinery is essential for engineering expanded genetic systems. Here we demonstrate that Escherichia coli RNA polymerase efficiently transcribes an eight-letter genetic alphabet with two orthogonal unnatural base pairs: P:Z and B:S pairs. To overcome G:Z misincorporation, we synthesize a higher-fidelity analogue, termed Z*, in which the C5 nitro group is replaced with a carboxamide. To elucidate substrate-recognition mechanisms, we determine four cryo-electron microscopy structures of RNA polymerase incorporating dZ:PTP or dP:Z*TP at 2.42-2.75 Å resolution. These structures, together with our early work on S:B pair, show that E. coli RNA polymerase is able to efficiently recognize these unnatural base pairs in the same manner as natural base pairs. Collectively, these results establish the feasibility of an eight-letter genetic alphabet for transcription.
History
DepositionDec 16, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2026Group: Data collection / Database references / Structure summary
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Revision 1.1Sep 2, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation_author / em_admin / em_author_list
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata
Item: _citation_author.name / _em_admin.last_update ..._citation_author.name / _em_admin.last_update / _em_admin.title / _em_author_list.author
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Revision 1.2Sep 16, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
G: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
N: Non-template strand DNA
R: RNA nonamer
T: Template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)382,12612
Polymers381,4448
Non-polymers6824
Water3,279182
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules AGIJK

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


Mass: 25526.039 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 158105.672 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8452.571 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#5: DNA chain Non-template strand DNA


Mass: 3407.249 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#7: DNA chain Template strand DNA


Mass: 6670.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules R

#6: RNA chain RNA nonamer


Mass: 2934.831 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 186 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-A1C49 / [[(2~{R},3~{R},4~{R},5~{S})-5-(5-aminocarbonyl-6-azanyl-2-oxidanylidene-1~{H}-pyridin-3-yl)-4-fluoranyl-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate


Mass: 527.184 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H17FN3O14P3 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 182 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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 RNA polymerase elongation complex containing the unnatural dP:Z*TP base pair in a trigger-loop-open conformation (b).
Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM software
IDNameVersionCategoryImage processing-ID
1cryoSPARCparticle selection1
2PHENIX1.21_5207model refinement
13cryoSPARC3D reconstruction1
14cryoSPARCparticle selection2
19cryoSPARC3D reconstruction2
20cryoSPARCparticle selection3
25cryoSPARC3D reconstruction3
Image processing
IDImage recording-ID
11
21
31
CTF correction
IDEM image processing-IDType
11NONE
22NONE
33NONE
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
12.64FSC 0.143 CUT-OFF5045719ZP2POINT
22.64FSC 0.143 CUT-OFF5045719ZP2POINT
32.64FSC 0.143 CUT-OFF5045719ZP2POINT
42.64FSC 0.143 CUT-OFF5045729ZP2POINT
52.64FSC 0.143 CUT-OFF5045729ZP2POINT
62.64FSC 0.143 CUT-OFF5045729ZP2POINT
72.64FSC 0.143 CUT-OFF5045739ZP2POINT
82.64FSC 0.143 CUT-OFF5045739ZP2POINT
92.64FSC 0.143 CUT-OFF5045739ZP2POINT
RefinementHighest resolution: 2.64 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00626238
ELECTRON MICROSCOPYf_angle_d0.69835608
ELECTRON MICROSCOPYf_dihedral_angle_d10.5483931
ELECTRON MICROSCOPYf_chiral_restr0.0484063
ELECTRON MICROSCOPYf_plane_restr0.0064534

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