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- PDB-9v4m: Crystal structure of the T.thermophilus transcription initiation ... -

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Basic information

Entry
Database: PDB / ID: 9v4m
TitleCrystal structure of the T.thermophilus transcription initiation complex bound to Ap4G
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template DNA Strand
  • RNA polymerase sigma factor SigA
  • Template DNA strand
KeywordsTRANSCRIPTION / Complex
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / 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. ...: / DNA-directed RNA polymerase subunit beta', hybrid domain / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsDuan, W. / Kaushik, A. / Serganov, A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM112904 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21GM151508 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM088118-09 United States
CitationJournal: Nat Chem Biol / Year: 2025
Title: Molecular basis for noncanonical transcription initiation from NpA alarmones.
Authors: Wenqian Duan / Abhishek Kaushik / Ilona C Unarta / Yue Wu / Mengjie M J Liu / Jacob W Weaver / Bing Wang / William J Rice / Daniel J Luciano / Joel G Belasco / Xuhui Huang / Evgeny Nudler / Alexander Serganov /
Abstract: Stress-induced dinucleoside tetraphosphates (NpNs, where N is adenosine, guanosine, cytosine or uridine) are ubiquitous in living organisms, yet their function has been largely elusive for over 50 ...Stress-induced dinucleoside tetraphosphates (NpNs, where N is adenosine, guanosine, cytosine or uridine) are ubiquitous in living organisms, yet their function has been largely elusive for over 50 years. Recent studies have revealed that RNA polymerase can influence the cellular lifetime of transcripts by incorporating these alarmones into RNA as 5'-terminal caps. Here we present structural and biochemical data that reveal the molecular basis of noncanonical transcription initiation from NpAs by Escherichia coli and Thermus thermophilus RNA polymerases. Our results show the influence of the first two nucleotide incorporation steps on capping efficiency and the different interactions of NpAs with transcription initiation complexes. These data provide critical insights into the substrate selectivity that dictates levels of Np capping in bacterial cells.
History
DepositionMay 24, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release
Revision 1.1Oct 29, 2025Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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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
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 SigA
G: Template DNA strand
H: Non-template DNA Strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,42517
Polymers444,1008
Non-polymers1,3259
Water1,20767
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area51780 Å2
ΔGint-226 kcal/mol
Surface area135590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)185.055, 103.211, 296.509
Angle α, β, γ (deg.)90.00, 98.89, 90.00
Int Tables number5
Space group name H-MC121
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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

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


Mass: 35056.164 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHR6, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125436.539 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 170997.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE8, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11533.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE7, DNA-directed RNA polymerase

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Protein , 1 types, 1 molecules F

#5: Protein RNA polymerase sigma factor SigA


Mass: 50900.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: sigA, TTHA0532 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SKW1

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DNA chain , 2 types, 2 molecules GH

#6: DNA chain Template DNA strand


Mass: 6698.319 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain Non-template DNA Strand


Mass: 8421.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 5 types, 76 molecules

#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: Mg
#9: Chemical ChemComp-A1IF9 / [[(2~{R},3~{S},4~{R},5~{R})-5-(6-aminopurin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl] [[[(2~{R},3~{S},4~{R},5~{R})-5-(2-azanyl-6-oxidanylidene-1~{H}-purin-9-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-oxidanyl-phosphoryl] hydrogen phosphate


Mass: 852.386 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28N10O20P4 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 67 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.15 Å3/Da / Density % sol: 60.95 %
Crystal growTemperature: 291.15 K / Method: vapor diffusion, hanging drop
Details: 180 mM Kcl, 50 mM Mgcl2, 12.5% PEG4000, 100 mM Tris.Hcl pH 8.7

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: 100 / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97895 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Jun 25, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97895 Å / Relative weight: 1
ReflectionResolution: 3→30 Å / Num. obs: 105883 / % possible obs: 95.6 % / Redundancy: 4.3 % / Biso Wilson estimate: 59.92 Å2 / CC1/2: 0.99 / CC star: 0.997 / Rmerge(I) obs: 0.126 / Rpim(I) all: 0.065 / Rrim(I) all: 0.143 / Net I/σ(I): 10.9
Reflection shellResolution: 3→3.11 Å / Redundancy: 4.1 % / Rmerge(I) obs: 1.24 / Num. unique obs: 10426 / CC1/2: 0.53 / CC star: 0.83 / % possible all: 28.9

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4Q5S

4q5s


Resolution: 3→29.96 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 32.1 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2884 2024 2.33 %
Rwork0.2791 --
obs0.2793 86969 78.39 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3→29.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms26463 755 76 67 27361
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00727890
X-RAY DIFFRACTIONf_angle_d0.90937891
X-RAY DIFFRACTIONf_dihedral_angle_d10.6924128
X-RAY DIFFRACTIONf_chiral_restr0.0624267
X-RAY DIFFRACTIONf_plane_restr0.0094831
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.070.3799500.36452086X-RAY DIFFRACTION27
3.07-3.160.3282730.34542955X-RAY DIFFRACTION38
3.16-3.250.38681090.34053954X-RAY DIFFRACTION52
3.25-3.350.34241030.32954756X-RAY DIFFRACTION62
3.35-3.470.34051360.33575467X-RAY DIFFRACTION71
3.47-3.610.31291460.32786178X-RAY DIFFRACTION80
3.61-3.780.30941610.31436877X-RAY DIFFRACTION89
3.78-3.980.35251840.29587192X-RAY DIFFRACTION94
3.98-4.220.31381680.2847388X-RAY DIFFRACTION95
4.22-4.550.24191730.2647446X-RAY DIFFRACTION96
4.55-50.26521740.26247504X-RAY DIFFRACTION97
5-5.720.30891800.2737522X-RAY DIFFRACTION97
5.72-7.20.31651800.27427776X-RAY DIFFRACTION99
7.2-29.960.20471870.22397844X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.8618-0.1573-0.21540.34140.20180.6964-0.01830.06070.08560.070.0419-0.0562-0.03170.33870.01160.4655-0.1689-0.06930.7633-0.0740.23818.86419.7679122.0885
20.74860.0299-0.08360.51650.00760.5732-0.0667-0.00770.1940.02990.10430.0391-0.3411-0.1076-0.04330.77320.02590.10560.2197-0.03860.3866-22.779422.7575127.6627
30.3223-0.0873-0.08590.31650.07570.4188-0.05620.0886-0.1253-0.04170.1018-0.07090.20041.0246-0.02640.36340.34640.00260.855-0.11340.3215.1991-12.70279.3235
40.4493-0.2612-0.11230.28330.00871.22180.13281.01330.0370.0084-0.17120.2379-0.13420.8410.06290.2394-0.1314-0.0543-0.0256-0.38530.2458-8.06653.047861.688
50.91660.48190.07080.97460.27530.0817-0.02580.06710.324-0.0244-0.0057-0.1127-0.28840.19560.03190.8772-0.2188-0.01460.352-0.08470.4438-12.017141.198982.2099
60.23160.0365-0.04050.20990.1050.21290.1150.14650.1578-0.0308-0.1402-0.0511-0.09970.0791-0.00330.56050.03030.01141.0959-0.03510.617230.870310.989740.8608
70.8717-0.2642-0.89511.6616-0.21851.06790.12120.2114-0.2851-0.13240.0917-0.09680.19920.2001-0.21380.4482-0.00710.07870.62-0.05890.6062-0.2107-4.331761.4032
80.6188-0.0289-0.5744-0.01080.02940.5344-0.0660.00560.0551-0.0135-0.0298-0.070.11780.14670.07930.95630.04130.00211.1241-0.03811.050313.0017-15.232242.9341
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain 'A' and resid 4 through 230)
2X-RAY DIFFRACTION2(chain 'B' and resid 4 through 229)
3X-RAY DIFFRACTION3(chain 'C' and resid 1 through 1118)
4X-RAY DIFFRACTION4(chain 'D' and resid 3 through 1499)
5X-RAY DIFFRACTION5(chain 'E' and resid 2 through 95)
6X-RAY DIFFRACTION6(chain 'F' and resid 77 through 423)
7X-RAY DIFFRACTION7(chain 'G' and resid 6 through 20)
8X-RAY DIFFRACTION8(chain 'H' and resid 1 through 22)

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