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- PDB-7q5b: Cryo-EM structure of Ty3 retrotransposon targeting a TFIIIB-bound... -

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Basic information

Entry
Database: PDB / ID: 7q5b
TitleCryo-EM structure of Ty3 retrotransposon targeting a TFIIIB-bound tRNA gene
Components
  • (Transcription factor ...) x 2
  • DNA (19-MER)
  • DNA (31-MER)
  • DNA (34-MER)
  • DNA (56-MER)
  • DNA (9-MER)
  • TATA-box-binding protein
  • Transposon Ty3-G Gag-Pol polyprotein
KeywordsDNA BINDING PROTEIN / Transcription / RNA Pol III / Ty3 Retrotransposon / Intasome
Function / homology
Function and homology information


retrotransposon nucleocapsid / RNA polymerase III core binding / retrotransposition / TFIIA-class transcription factor complex binding / transcription factor TFIIIB complex / RNA polymerase III preinitiation complex assembly / RNA polymerase III transcription regulatory region sequence-specific DNA binding / DNA-templated transcription open complex formation / TFIIIC-class transcription factor complex binding / RNA polymerase III type 3 promoter sequence-specific DNA binding ...retrotransposon nucleocapsid / RNA polymerase III core binding / retrotransposition / TFIIA-class transcription factor complex binding / transcription factor TFIIIB complex / RNA polymerase III preinitiation complex assembly / RNA polymerase III transcription regulatory region sequence-specific DNA binding / DNA-templated transcription open complex formation / TFIIIC-class transcription factor complex binding / RNA polymerase III type 3 promoter sequence-specific DNA binding / regulation of transcription by RNA polymerase III / RNA polymerase I general transcription initiation factor binding / RNA polymerase III general transcription initiation factor activity / transcription factor TFIIA complex / RNA polymerase I preinitiation complex assembly / transcription preinitiation complex / DNA binding, bending / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / RNA polymerase II transcribes snRNA genes / RNA Polymerase I Promoter Escape / Estrogen-dependent gene expression / RNA polymerase II general transcription initiation factor activity / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / transcription factor TFIID complex / RNA Polymerase II Pre-transcription Events / nucleolar large rRNA transcription by RNA polymerase I / RNA polymerase II core promoter sequence-specific DNA binding / RNA nuclease activity / transcription by RNA polymerase III / RNA polymerase II preinitiation complex assembly / TBP-class protein binding / DNA-templated transcription initiation / DNA integration / RNA-directed DNA polymerase / disordered domain specific binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / peptidase activity / DNA-binding transcription factor binding / DNA recombination / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / negative regulation of DNA-templated transcription / chromatin binding / regulation of DNA-templated transcription / positive regulation of transcription by RNA polymerase II / protein-containing complex / proteolysis / DNA binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Peptidase A2B, Ty3 transposon peptidase / Ty3 transposon peptidase / Ty3 transposon capsid-like protein / Ty3 transposon capsid-like protein / Transcription factor TFIIIB component B'', fungi / Brf1, TBP-binding domain / Brf1-like TBP-binding domain / Transcription factor TFIIIB component B'', Myb domain / Myb DNA-binding like / Integrase zinc-binding domain ...Peptidase A2B, Ty3 transposon peptidase / Ty3 transposon peptidase / Ty3 transposon capsid-like protein / Ty3 transposon capsid-like protein / Transcription factor TFIIIB component B'', fungi / Brf1, TBP-binding domain / Brf1-like TBP-binding domain / Transcription factor TFIIIB component B'', Myb domain / Myb DNA-binding like / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / Transcription factor TFIIB, cyclin-like domain / Transcription factor TFIIB, conserved site / Transcription factor TFIIB repeat / Transcription factor TFIIB repeat signature. / Transcription factor TFIIB / Zinc finger TFIIB-type profile. / Zinc finger, TFIIB-type / TFIIB zinc-binding / TATA-box binding protein, eukaryotic / TATA-box binding protein / TATA-box binding protein, conserved site / Transcription factor TFIID (or TATA-binding protein, TBP) / Transcription factor TFIID repeat signature. / TBP domain superfamily / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / Homeobox-like domain superfamily / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / zinc finger / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / TATA-box-binding protein / Transcription factor IIIB 70 kDa subunit / Transcription factor TFIIIB component B'' / Transposon Ty3-G Gag-Pol polyprotein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.98 Å
AuthorsAbascal-Palacios, G. / Jochem, L. / Pla-Prats, C. / Beuron, F. / Vannini, A.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Cancer Research UKCR-UK C47547/A21536 United Kingdom
Wellcome Trust200818/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes.
Authors: Guillermo Abascal-Palacios / Laura Jochem / Carlos Pla-Prats / Fabienne Beuron / Alessandro Vannini /
Abstract: Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity ...Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.
History
DepositionNov 3, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
R: DNA (56-MER)
S: DNA (31-MER)
r: DNA (34-MER)
s: DNA (9-MER)
A: Transposon Ty3-G Gag-Pol polyprotein
B: Transposon Ty3-G Gag-Pol polyprotein
C: Transposon Ty3-G Gag-Pol polyprotein
D: Transposon Ty3-G Gag-Pol polyprotein
X: Transcription factor TFIIIB component B''
Y: TATA-box-binding protein
Z: Transcription factor IIIB 70 kDa subunit
t: DNA (19-MER)
u: DNA (19-MER)


Theoretical massNumber of molelcules
Total (without water)927,77913
Polymers927,77913
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Superose 6 increase 10/300 GL column (GE Healthcare), native gel electrophoresis, Electrophoretic Mobility Shift Assay (EMSA)
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "u"
d_2ens_1chain "t"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-ID
d_11ens_1DGDCM
d_21ens_1DGDCL

NCS oper: (Code: givenMatrix: (-0.98414504866, -0.00966245960061, 0.17710211764), (0.0285295605382, -0.994136793859, 0.104298126878), (0.175055954979, 0.107697130738, 0.978650469094)Vector: 297. ...NCS oper: (Code: given
Matrix: (-0.98414504866, -0.00966245960061, 0.17710211764), (0.0285295605382, -0.994136793859, 0.104298126878), (0.175055954979, 0.107697130738, 0.978650469094)
Vector: 297.246593967, 307.092016465, -42.674799968)

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Components

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DNA chain , 5 types, 6 molecules RSrstu

#1: DNA chain DNA (56-MER)


Mass: 17165.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#2: DNA chain DNA (31-MER)


Mass: 9484.193 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#3: DNA chain DNA (34-MER)


Mass: 10532.895 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#4: DNA chain DNA (9-MER)


Mass: 2751.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#9: DNA chain DNA (19-MER)


Mass: 5882.792 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)

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Protein , 2 types, 5 molecules ABCDY

#5: Protein
Transposon Ty3-G Gag-Pol polyprotein / Gag3-Pol3 / Transposon Ty3-1 TYA-TYB polyprotein


Mass: 178556.016 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Escherichia coli (E. coli) / References: UniProt: Q99315
#7: Protein TATA-box-binding protein / TATA sequence-binding protein / TBP / TATA-binding factor / TATA-box factor / Transcription factor ...TATA sequence-binding protein / TBP / TATA-binding factor / TATA-box factor / Transcription factor D / Transcription initiation factor TFIID TBP subunit


Mass: 27042.275 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Escherichia coli (E. coli) / References: UniProt: P13393

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Transcription factor ... , 2 types, 2 molecules XZ

#6: Protein Transcription factor TFIIIB component B'' / TFIIIB90


Mass: 67801.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Escherichia coli (E. coli) / References: UniProt: P46678
#8: Protein Transcription factor IIIB 70 kDa subunit / TFIIIB / B-related factor 1 / BRF-1


Mass: 67011.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: BRF1, PCF4, TDS4, YGR246C / Production host: Escherichia coli (E. coli) / References: UniProt: P29056

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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

Component
IDNameTypeEntity IDParent-IDSource
1Ty3 retrotransposon engaged with TFIIIB transcription factor and tRNA promoterCOMPLEXall0MULTIPLE SOURCES
2DNACOMPLEX#1-#4, #91RECOMBINANT
3TFIIIB transcription factorCOMPLEX#5-#81RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae S288C (yeast)559292
23Saccharomyces cerevisiae S288C (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
140 mMTris-HClTris1
280 mMNaClSodium chloride1
37 mMMgCl21
41 mMDTT1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: Glow discharged at 15 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELDBright-field microscopy / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDNominal defocus max (nm)Nominal defocus min (nm)Nominal magnification (X)
132001700130000
24000240081000
Image recording
IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real images
11750GATAN K2 SUMMIT (4k x 4k)14974
225.370GATAN K3 (6k x 4k)12437

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.1_3865refinement
PHENIX1.18.1_3865refinement
EM software
IDNameVersionCategoryImaging-ID
2EPUimage acquisition1
4CTFFIND4CTF correction
9EPUimage acquisition2
10RELION3.0.4initial Euler assignment
11RELION3.0.4final Euler assignment
12RELION3.0.4classification
13RELION3.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 628998
3D reconstructionResolution: 3.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101469 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Correlation Coefficient
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 85.1 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004222550
ELECTRON MICROSCOPYf_angle_d0.852931213
ELECTRON MICROSCOPYf_chiral_restr0.05233516
ELECTRON MICROSCOPYf_plane_restr0.00563378
ELECTRON MICROSCOPYf_dihedral_angle_d30.64764145
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.000712025323096 Å

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