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- PDB-7uik: Mediator-PIC Early (Tail A + Upstream DNA & Activator) -

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Basic information

Entry
Database: PDB / ID: 7uik
TitleMediator-PIC Early (Tail A + Upstream DNA & Activator)
Components
  • (Mediator of RNA polymerase II transcription subunit ...) x 6
  • DNA (37-MER)
  • DNA (38-MER)
  • Regulatory protein GAL4
KeywordsTRANSCRIPTION / Divergent transcription / Mediator / RNA Polymerase II / PIC / Pre-Initiation / Activation / Transcription Factor / Gal4 / Gal4VP16
Function / homology
Function and homology information


regulation of transcription from RNA polymerase II promoter by galactose / TFIIE-class transcription factor complex binding / regulation of establishment of protein localization to chromosome / positive regulation of invasive growth in response to glucose limitation / TFIIH-class transcription factor complex binding / core mediator complex / galactose metabolic process / mediator complex / positive regulation of transcription from RNA polymerase II promoter by galactose / TFIIB-class transcription factor binding ...regulation of transcription from RNA polymerase II promoter by galactose / TFIIE-class transcription factor complex binding / regulation of establishment of protein localization to chromosome / positive regulation of invasive growth in response to glucose limitation / TFIIH-class transcription factor complex binding / core mediator complex / galactose metabolic process / mediator complex / positive regulation of transcription from RNA polymerase II promoter by galactose / TFIIB-class transcription factor binding / positive regulation of transcription initiation by RNA polymerase II / RNA polymerase II core promoter sequence-specific DNA binding / RNA polymerase II preinitiation complex assembly / transcription repressor complex / positive regulation of transcription elongation by RNA polymerase II / transcription coregulator activity / DNA-binding transcription activator activity, RNA polymerase II-specific / RNA polymerase II-specific DNA-binding transcription factor binding / transcription by RNA polymerase II / transcription coactivator activity / DNA-binding transcription factor activity, RNA polymerase II-specific / RNA polymerase II cis-regulatory region sequence-specific DNA binding / DNA-templated transcription / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / zinc ion binding / identical protein binding / nucleus / cytosol
Similarity search - Function
Mediator of RNA polymerase II transcription subunit 3 / Mediator complex, subunit Med2, fungi / Mediator complex subunit 2 / Mediator complex subunit 3 fungal / Mediator complex, subunit Med5, fungi / Mediator complex subunit Med5 / Gal4 dimerisation domain / Gal4-like dimerisation domain / Mediator complex, subunit Med15, fungi / Gal11, coactivator domain ...Mediator of RNA polymerase II transcription subunit 3 / Mediator complex, subunit Med2, fungi / Mediator complex subunit 2 / Mediator complex subunit 3 fungal / Mediator complex, subunit Med5, fungi / Mediator complex subunit Med5 / Gal4 dimerisation domain / Gal4-like dimerisation domain / Mediator complex, subunit Med15, fungi / Gal11, coactivator domain / Mediator complex subunit 15, KIX domain / Mediator complex subunit 15 / KIX domain / Gal11 activator-binding domain (ABD1) / Fungal specific transcription factor domain / Transcription factor domain, fungi / Fungal specific transcription factor domain / : / : / Mediator complex subunit 16, C-terminal / Mediator complex, subunit Med16 / Mediator complex subunit 16, N-terminal / Mediator complex, subunit Med14 / Mediator complex subunit MED14 / Zn(2)-C6 fungal-type DNA-binding domain signature. / Fungal Zn(2)-Cys(6) binuclear cluster domain / Zn(2)-C6 fungal-type DNA-binding domain superfamily / Zn(2)-C6 fungal-type DNA-binding domain profile. / GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA-binding domain / Zn(2)-C6 fungal-type DNA-binding domain / Coactivator CBP, KIX domain superfamily / Basic-leucine zipper domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Regulatory protein GAL4 / Mediator of RNA polymerase II transcription subunit 14 / Mediator of RNA polymerase II transcription subunit 15 / Mediator of RNA polymerase II transcription subunit 16 / Mediator of RNA polymerase II transcription subunit 3 / Mediator of RNA polymerase II transcription subunit 5 / Mediator of RNA polymerase II transcription subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.7 Å
AuthorsGorbea Colon, J.J. / Chen, S.-F. / Tsai, K.L. / Murakami, K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM123233-01 United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of a transcription pre-initiation complex on a divergent promoter.
Authors: Jose J Gorbea Colón / Leon Palao / Shin-Fu Chen / Hee Jong Kim / Laura Snyder / Yi-Wei Chang / Kuang-Lei Tsai / Kenji Murakami /
Abstract: Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single ...Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.
History
DepositionMar 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
X: DNA (38-MER)
Y: DNA (37-MER)
T: Regulatory protein GAL4
U: Regulatory protein GAL4
o: Mediator of RNA polymerase II transcription subunit 15
b: Mediator of RNA polymerase II transcription subunit 2
c: Mediator of RNA polymerase II transcription subunit 3
n: Mediator of RNA polymerase II transcription subunit 14
e: Mediator of RNA polymerase II transcription subunit 5
p: Mediator of RNA polymerase II transcription subunit 16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)536,32414
Polymers536,06210
Non-polymers2624
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules XY

#1: DNA chain DNA (38-MER)


Mass: 11666.479 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (37-MER)


Mass: 11424.310 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli (E. coli)

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Mediator of RNA polymerase II transcription subunit ... , 6 types, 6 molecules obcnep

#4: Protein Mediator of RNA polymerase II transcription subunit 15 / Autonomous replication regulatory protein 3 / Basal expression activator protein 1 / Defective ...Autonomous replication regulatory protein 3 / Basal expression activator protein 1 / Defective silencing suppressor protein 4 / Mediator complex subunit 15 / Transcription regulatory protein GAL11 / Ty insertion suppressor protein 13


Mass: 120432.242 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / References: UniProt: P19659
#5: Protein Mediator of RNA polymerase II transcription subunit 2 / Mediator complex subunit 2


Mass: 47754.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / References: UniProt: Q12124
#6: Protein Mediator of RNA polymerase II transcription subunit 3 / Hyper-recombination suppressor protein 1 / Mediator complex subunit 3 / Poly-glutamine domain protein 1


Mass: 43116.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P40356
#7: Protein Mediator of RNA polymerase II transcription subunit 14 / Glucose repression regulatory protein 1 / Mediator complex subunit 14


Mass: 27535.459 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P19263
#8: Protein Mediator of RNA polymerase II transcription subunit 5 / Mediator complex subunit 5 / Negative regulator of URS2 protein 1


Mass: 128918.750 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / References: UniProt: P53114
#9: Protein Mediator of RNA polymerase II transcription subunit 16 / Global transcriptional regulator SIN4 / Mediator complex subunit 16 / SNF1 suppressor protein 4 / ...Global transcriptional regulator SIN4 / Mediator complex subunit 16 / SNF1 suppressor protein 4 / SWI4 suppressor protein 5 / Transcriptional silencing factor 3 / YGP1 expression regulatory protein 1


Mass: 111423.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / References: UniProt: P32259

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Protein / Non-polymers , 2 types, 6 molecules TU

#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Protein Regulatory protein GAL4


Mass: 16895.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: GAL4, YPL248C / Production host: Escherichia coli (E. coli) / References: UniProt: P04386

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Details

Has ligand of interestY

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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: Med-PICEarly (TailA+Activator & DNA Class 1) / Type: COMPLEX / Entity ID: #1-#2, #4-#6, #3, #8-#9 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMPotassium acetateKOAc1
220 mMHEPESHEPES1
35 mMDithiothreitolDTT1
42 mMMagnesium acetateMg(OAc)21
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Calibrated magnification: 64000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.4 sec. / Electron dose: 42 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 40900

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199834 / Symmetry type: POINT

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