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- PDB-7ubj: Transcription antitermination factor Qlambda, type-I crystal -

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Basic information

Entry
Database: PDB / ID: 7ubj
TitleTranscription antitermination factor Qlambda, type-I crystal
ComponentsAntitermination protein Q
KeywordsGENE REGULATION / RNA polymerase / DNA Binding / transcription / Q-dependent antitermination / Q antitermination factor
Function / homology
Function and homology information


transcription antitermination / DNA-templated transcription termination / DNA binding / zinc ion binding
Similarity search - Function
Enzyme I; Chain A, domain 2 - #110 / Antitermination protein Q / Antitermination protein Q superfamily / Antitermination protein Q / Heat shock protein DnaJ, cysteine-rich domain superfamily / Enzyme I; Chain A, domain 2 / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / PHOSPHATE ION / Antitermination protein Q
Similarity search - Component
Biological speciesEscherichia phage Lambda (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.46 Å
AuthorsYin, Z. / Ebright, R.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)GM041376 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.
Authors: Zhou Yin / Jeremy G Bird / Jason T Kaelber / Bryce E Nickels / Richard H Ebright /
Abstract: Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP ...Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.
History
DepositionMar 15, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 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: Antitermination protein Q
B: Antitermination protein Q
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,59118
Polymers32,4462
Non-polymers1,14516
Water8,017445
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3710 Å2
ΔGint-21 kcal/mol
Surface area17800 Å2
Unit cell
Length a, b, c (Å)54.341, 54.341, 110.090
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number145
Space group name H-MP32

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Antitermination protein Q


Mass: 16222.834 Da / Num. of mol.: 2 / Fragment: UNP residues 62-207 / Mutation: E134K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage Lambda (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: P03047

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Non-polymers , 7 types, 461 molecules

#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H6O2
#6: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H10O3
#7: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 445 / Source method: isolated from a natural source / Formula: H2O

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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: 2.89 Å3/Da / Density % sol: 57.47 %
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 20% v/v glycerol, 0.4 M potassium phosphate dibasic, 16% PEG8000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.987 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 5, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 1.46→50 Å / Num. obs: 63011 / % possible obs: 99.7 % / Redundancy: 5.1 % / Rmerge(I) obs: 0.049 / Rpim(I) all: 0.024 / Rrim(I) all: 0.055 / Χ2: 0.85 / Net I/σ(I): 11.9 / Num. measured all: 319426
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.46-1.495.10.88731160.650.4380.9910.845100
1.49-1.515.10.66132090.7570.3270.7390.832100
1.51-1.5450.57331320.7940.2850.6420.86499.9
1.54-1.5750.45431780.8690.2260.5080.86199.9
1.57-1.614.80.37131190.9030.1870.4170.87899.8
1.61-1.644.70.31131680.9120.1590.350.8898.4
1.64-1.695.20.26131340.950.1270.290.895100
1.69-1.735.20.20731600.9630.1010.230.88299.9
1.73-1.785.20.16131330.9780.0790.180.884100
1.78-1.845.10.13231760.980.0650.1470.999.9
1.84-1.914.90.10631380.9860.0530.1190.90699.8
1.91-1.984.80.0931250.9880.0450.10.90598.8
1.98-2.075.30.07631430.9920.0370.0850.895100
2.07-2.185.20.06932120.9930.0330.0770.922100
2.18-2.325.20.0631250.9940.0290.0670.86699.8
2.32-2.54.80.05431480.9940.0270.0610.78299.4
2.5-2.755.40.05331600.9950.0250.0590.78399.9
2.75-3.155.30.04931560.9950.0240.0540.718100
3.15-3.964.90.04331310.9950.0220.0480.76398.9
3.96-505.20.03731480.9960.0180.0410.74399.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
PHENIX1.14_3260refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4MO1

4mo1


Resolution: 1.46→43.273 Å / SU ML: 0.14 / Cross valid method: THROUGHOUT / σ(F): 1.98 / Phase error: 22.63 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2026 3278 3.36 %
Rwork0.1775 94366 -
obs0.1783 59450 77.33 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 88.93 Å2 / Biso mean: 23.5208 Å2 / Biso min: 4.42 Å2
Refinement stepCycle: final / Resolution: 1.46→43.273 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2231 0 139 445 2815
Biso mean--39.36 31.84 -
Num. residues----290
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.46-1.48180.2937390.2948105820
1.4818-1.50490.2874520.2699162231
1.5049-1.52960.306700.2623213740
1.5296-1.5560.2821970.2531252147
1.556-1.58430.258880.2426257749
1.5843-1.61480.2742890.2307267350
1.6148-1.64770.24351070.2211284953
1.6477-1.68360.18971190.2065319861
1.6836-1.72270.2121290.197372571
1.7227-1.76580.21751430.2025439083
1.7658-1.81350.20461770.2007494792
1.8135-1.86690.26451720.2028512796
1.8669-1.92720.20261780.2064505796
1.9272-1.9960.21661840.1902516998
1.996-2.0760.2111840.17885364100
2.076-2.17040.20981770.1787524199
2.1704-2.28490.15281670.1678528999
2.2849-2.4280.21391880.168522099
2.428-2.61550.19841750.1673529998
2.6155-2.87860.19621830.17345260100
2.8786-3.2950.19781960.17275235100
3.295-4.15090.19231850.1452521697
4.1509-43.2730.18111790.1652519298

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