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- PDB-9bdd: Cryo-EM Structure of Non-Cognate Substrate Bound in the Entry Sit... -

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Basic information

Entry
Database: PDB / ID: 9bdd
TitleCryo-EM Structure of Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Components
  • DNA-directed RNA polymerase, mitochondrial
  • Non-Template Strand DNA (NT27mt_+1T)
  • RNA (RNA14mt)
  • Template Strand DNA (TS31mt_+1A)
  • Transcription elongation factor, mitochondrial
KeywordsTRANSCRIPTION/DNA/RNA / Mitochondrial RNA Polymerase / Nucleotide Selection / Nucleotide Discrimination / TRANSCRIPTION / Protein-RNA-DNA Complex / POLRMT / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


transcription elongation by mitochondrial RNA polymerase / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / mitochondrial transcription / DNA primase activity / oxidative phosphorylation / DNA polymerase processivity factor activity / mitochondrial nucleoid ...transcription elongation by mitochondrial RNA polymerase / Mitochondrial transcription initiation / mitochondrial DNA-directed RNA polymerase complex / mitochondrial promoter sequence-specific DNA binding / transcription initiation at mitochondrial promoter / mitochondrial transcription / DNA primase activity / oxidative phosphorylation / DNA polymerase processivity factor activity / mitochondrial nucleoid / Transcriptional activation of mitochondrial biogenesis / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 3'-5'-RNA exonuclease activity / sequence-specific DNA binding / mitochondrial matrix / ribonucleoprotein complex / protein-containing complex / mitochondrion / RNA binding
Similarity search - Function
Transcription elongation factor, mitochondrial / Helix-hairpin-helix motif / DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase ...Transcription elongation factor, mitochondrial / Helix-hairpin-helix motif / DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase / Bacteriophage-type RNA polymerase family active site signature 2. / RuvA domain 2-like / Tetratricopeptide-like helical domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase, mitochondrial / Transcription elongation factor, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsHerbine, K.H. / Nayak, A.R. / Temiakov, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM131832 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for substrate binding and selection by human mitochondrial RNA polymerase.
Authors: Karl Herbine / Ashok R Nayak / Dmitry Temiakov /
Abstract: The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM ...The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.
History
DepositionApr 11, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transcription elongation factor, mitochondrial
B: Transcription elongation factor, mitochondrial
E: DNA-directed RNA polymerase, mitochondrial
N: Non-Template Strand DNA (NT27mt_+1T)
R: RNA (RNA14mt)
T: Template Strand DNA (TS31mt_+1A)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,5777
Polymers207,0726
Non-polymers5051
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules ABE

#1: Protein Transcription elongation factor, mitochondrial


Mass: 27262.389 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TEFM, C17orf42 / Production host: Escherichia coli (E. coli) / References: UniProt: Q96QE5
#2: Protein DNA-directed RNA polymerase, mitochondrial


Mass: 127150.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLRMT / Production host: Escherichia coli (E. coli) / References: UniProt: O00411

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DNA chain , 2 types, 2 molecules NT

#3: DNA chain Non-Template Strand DNA (NT27mt_+1T)


Mass: 10474.738 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: DNA chain Template Strand DNA (TS31mt_+1A)


Mass: 10428.664 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain / Non-polymers , 2 types, 2 molecules R

#4: RNA chain RNA (RNA14mt)


Mass: 4493.723 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: Chemical ChemComp-APC / DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / ALPHA,BETA-METHYLENEADENOSINE-5'-TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-CPP, energy-carrying molecule analogue*YM

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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: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Type: COMPLEX
Details: Human Mitochondrial RNA polymerase (d119) and Human TEFM (d50) assembled on an RNA-DNA Scaffold in presence of methylene a,b-ATP.
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.227 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Details: 20 mM Tris Buffer, pH 7.9, 150 mM NaCl, 10 mM DTT, and 5 mM MgCl2
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 5 uM complex of (D119) wild-type mtRNAP, (D50) wild-type TEFM, an RNA-DNA scaffold (R14/T31_+1A/NT27_+1T), and a,b methylene ATP.
Specimen supportDetails: 15 mA Discharge / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Preliminary grid screening performed manually using TFS Glacios.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.89 sec. / Electron dose: 50.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 21764
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_5015: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7998459
Details: Automated particle picking (Blob picker, CryoSPARC) with manual inspection (Inspect Particle Picks).
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 497782
Details: Non-uniform Refinement (CryoSPARC) which is an algorithm based on cross-validation optimization, which automatically regularizes 3D density maps during refinement to account for spatial variability.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 45 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation Coefficient
Details: Initial Fitting was done in Chimera and flexible/refined fitting done with Coot Phenix Real-Space Refinement.
Atomic model buildingPDB-ID: 5OLA

5ola


Accession code: 5OLA / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312722
ELECTRON MICROSCOPYf_angle_d0.61817530
ELECTRON MICROSCOPYf_dihedral_angle_d16.7412139
ELECTRON MICROSCOPYf_chiral_restr0.0411987
ELECTRON MICROSCOPYf_plane_restr0.0062021

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