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- PDB-7mlb: Crystal structure of Thermus thermophilus transcription initiatio... -

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Basic information

Entry
Database: PDB / ID: 7mlb
TitleCrystal structure of Thermus thermophilus transcription initiation complex with 5nt RNA
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (5'-D(*TP*AP*TP*AP*AP*TP*GP*GP*AP*GP*GP*GP*AP*TP*GP*CP*AP*G)-3')
  • DNA (5'-D(P*CP*TP*GP*CP*AP*TP*CP*CP*CP*TP*GP*AP*GP*TP*G)-3')
  • RNA (5'-R(P*AP*CP*UP*CP*A)-3')
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION / Thermus thermophilus / transcription initiation
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 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 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 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, 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 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / 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 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, 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 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-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / 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 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.6 Å
AuthorsLiu, Y. / Ebright, R.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structural and mechanistic basis of reiterative transcription initiation.
Authors: Yu Liu / Libing Yu / Chirangini Pukhrambam / Jared T Winkelman / Emre Firlar / Jason T Kaelber / Yu Zhang / Bryce E Nickels / Richard H Ebright /
Abstract: Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the ...Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the DNA template. Here, using crystallography and cryoelectron microscopy to define structures, protein-DNA photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNA polymerase (RNAP)-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation 1) occurs without DNA scrunching; 2) involves a short, 2- to 3-bp, RNA-DNA hybrid; and 3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit.
History
DepositionApr 28, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: DNA (5'-D(P*CP*TP*GP*CP*AP*TP*CP*CP*CP*TP*GP*AP*GP*TP*G)-3')
H: DNA (5'-D(*TP*AP*TP*AP*AP*TP*GP*GP*AP*GP*GP*GP*AP*TP*GP*CP*AP*G)-3')
I: RNA (5'-R(P*AP*CP*UP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,57316
Polymers445,3219
Non-polymers2527
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area52850 Å2
ΔGint-245 kcal/mol
Surface area141220 Å2
MethodPISA
Unit cell
Length a, b, c (Å)186.172, 104.266, 299.017
Angle α, β, γ (deg.)90.000, 97.949, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / 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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / 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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / 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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / References: UniProt: Q8RQE7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules GH

#6: DNA chain DNA (5'-D(P*CP*TP*GP*CP*AP*TP*CP*CP*CP*TP*GP*AP*GP*TP*G)-3')


Mass: 6480.172 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (5'-D(*TP*AP*TP*AP*AP*TP*GP*GP*AP*GP*GP*GP*AP*TP*GP*CP*AP*G)-3')


Mass: 8461.456 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules FI

#5: Protein RNA polymerase sigma factor SigA


Mass: 50769.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: sigA, TTHA0532 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SKW1
#8: RNA chain RNA (5'-R(P*AP*CP*UP*CP*A)-3')


Mass: 1529.984 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 7 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: 3.24 Å3/Da / Density % sol: 62.07 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8.4
Details: 0.1 M Tris-HCl pH8.4, 180 mM potassium chloride, 50 mM Magnesium chloride, 9% (m/v) PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.97907 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 6, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97907 Å / Relative weight: 1
ReflectionResolution: 3.6→50 Å / Num. obs: 66110 / % possible obs: 98.5 % / Redundancy: 4 % / Biso Wilson estimate: 116.36 Å2 / Rmerge(I) obs: 0.116 / Rsym value: 0.116 / Net I/σ(I): 10.733
Reflection shellResolution: 3.6→3.66 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.845 / Num. unique obs: 3055 / CC1/2: 0.624 / Rsym value: 0.845 / % possible all: 92.3

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4G7H

4g7h


Resolution: 3.6→44.62 Å / SU ML: 0.4961 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 30.8326
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2697 1998 3.07 %
Rwork0.233 63039 -
obs0.2341 65037 98.32 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 144.6 Å2
Refinement stepCycle: LAST / Resolution: 3.6→44.62 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27608 785 7 0 28400
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002228982
X-RAY DIFFRACTIONf_angle_d0.534539357
X-RAY DIFFRACTIONf_chiral_restr0.04174436
X-RAY DIFFRACTIONf_plane_restr0.00435029
X-RAY DIFFRACTIONf_dihedral_angle_d14.804511258
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.6-3.690.37111370.31984209X-RAY DIFFRACTION92.31
3.69-3.790.36771400.30524375X-RAY DIFFRACTION96.31
3.79-3.90.32031500.28814472X-RAY DIFFRACTION99.21
3.9-4.030.3241270.27614542X-RAY DIFFRACTION99.38
4.03-4.170.27991490.26274531X-RAY DIFFRACTION99.55
4.17-4.340.34271410.24974563X-RAY DIFFRACTION99.56
4.34-4.530.28021510.24584460X-RAY DIFFRACTION98.5
4.54-4.770.28931380.23264504X-RAY DIFFRACTION98.6
4.77-5.070.27541500.22534573X-RAY DIFFRACTION99.7
5.07-5.460.25251460.2264541X-RAY DIFFRACTION99.62
5.46-6.010.27341430.23514531X-RAY DIFFRACTION99.03
6.01-6.880.24621380.2334553X-RAY DIFFRACTION98.78
6.88-8.660.23531500.21334619X-RAY DIFFRACTION99.56
8.66-44.620.22521380.1964566X-RAY DIFFRACTION96.53

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