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- EMDB-44449: Cryo-EM Structure of Non-Cognate Substrate Bound in the Entry Sit... -

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Entry
Database: EMDB / ID: EMD-44449
TitleCryo-EM Structure of Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Map dataStructure of Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Sample
  • Complex: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
    • Protein or peptide: Transcription elongation factor, mitochondrial
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • DNA: Non-Template Strand DNA (NT27mt_+1T)
    • RNA: RNA (RNA14mt)
    • DNA: Template Strand DNA (TS31mt_+1A)
  • Ligand: DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER
KeywordsMitochondrial 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
DNA-directed RNA polymerase, mitochondrial / Transcription elongation factor, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsHerbine KH / Nayak AR / Temiakov D
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44449.png

Downloads & links

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Map

FileDownload / File: emd_44449.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 296 pix.
= 247.101 Å		0.83 Å/pix.
x 296 pix.
= 247.101 Å		0.83 Å/pix.
x 296 pix.
= 247.101 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8348 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0281
Minimum - Maximum-0.025855279 - 2.0041122
Average (Standard dev.)0.0015520257 (±0.02796201)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 247.1008 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_44449_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_44449_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitoc...

EntireName: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
Components
  • Complex: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
    • Protein or peptide: Transcription elongation factor, mitochondrial
    • Protein or peptide: DNA-directed RNA polymerase, mitochondrial
    • DNA: Non-Template Strand DNA (NT27mt_+1T)
    • RNA: RNA (RNA14mt)
    • DNA: Template Strand DNA (TS31mt_+1A)
  • Ligand: DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER

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Supramolecule #1: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitoc...

SupramoleculeName: Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: Human Mitochondrial RNA polymerase (d119) and Human TEFM (d50) assembled on an RNA-DNA Scaffold in presence of methylene a,b-ATP.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 227 KDa

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Macromolecule #1: Transcription elongation factor, mitochondrial

MacromoleculeName: Transcription elongation factor, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.262389 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDENAKEPEN RFLRKLLKPD IERERLKAVN SIISIVFGTR RIAWAHLDRK LTVLDWQQSD RWSLMRGIYS SSVYLEEISS IISKMPKAD FYVLEKTGLS IQNSSLFPIL LHFHIMEAML YALLNKTFAQ DGQHQVLSMN RNAVGKHFEL MIGDSRTSGK E LVKQFLFD ...String:
MDENAKEPEN RFLRKLLKPD IERERLKAVN SIISIVFGTR RIAWAHLDRK LTVLDWQQSD RWSLMRGIYS SSVYLEEISS IISKMPKAD FYVLEKTGLS IQNSSLFPIL LHFHIMEAML YALLNKTFAQ DGQHQVLSMN RNAVGKHFEL MIGDSRTSGK E LVKQFLFD SILKADPRVF FPSDKIVHYR QMFLSTELQR VEELYDSLLQ AIAFYELAVF DSQPLEHHHH HHHH

UniProtKB: Transcription elongation factor, mitochondrial

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Macromolecule #2: DNA-directed RNA polymerase, mitochondrial

MacromoleculeName: DNA-directed RNA polymerase, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 127.150102 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHGR WAKILEKDKR TQQMRMQRLK AKLQMPFQSG EFKALTRRLQ VEPRLLSKQM AGCLEDCTRQ APESPWEEQL ARLLQEAPG KLSLDVEQAP SGQHSQAQLS GQQQRLLAFF KCCLLTDQLP LAHHLLVVHH GQRQKRKLLT LDMYNAVMLG W ARQGAFKE ...String:
MGHHHHHHGR WAKILEKDKR TQQMRMQRLK AKLQMPFQSG EFKALTRRLQ VEPRLLSKQM AGCLEDCTRQ APESPWEEQL ARLLQEAPG KLSLDVEQAP SGQHSQAQLS GQQQRLLAFF KCCLLTDQLP LAHHLLVVHH GQRQKRKLLT LDMYNAVMLG W ARQGAFKE LVYVLFMVKD AGLTPDLLSY AAALQCMGRQ DQDAGTIERC LEQMSQEGLK LQALFTAVLL SEEDRATVLK AV HKVKPTF SLPPQLPPPV NTSKLLRDVY AKDGRVSYPK LHLPLKTLQC LFEKQLHMEL ASRVCVVSVE KPTLPSKEVK HAR KTLKTL RDQWEKALCR ALRETKNRLE REVYEGRFSL YPFLCLLDER EVVRMLLQVL QALPAQGESF TTLARELSAR TFSR HVVQR QRVSGQVQAL QNHYRKYLCL LASDAEVPEP CLPRQYWEAL GAPEALREQP WPLPVQMELG KLLAEMLVQA TQMPC SLDK PHRSSRLVPV LYHVYSFRNV QQIGILKPHP AYVQLLEKAA EPTLTFEAVD VPMLCPPLPW TSPHSGAFLL SPTKLM RTV EGATQHQELL ETCPPTALHG ALDALTQLGN CAWRVNGRVL DLVLQLFQAK GCPQLGVPAP PSEAPQPPEA HLPHSAA PA RKAELRRELA HCQKVAREMH SLRAEALYRL SLAQHLRDRV FWLPHNMDFR GRTYPCPPHF NHLGSDVARA LLEFAQGR P LGPHGLDWLK IHLVNLTGLK KREPLRKRLA FAEEVMDDIL DSADQPLTGR KWWMGAEEPW QTLACCMEVA NAVRASDPA AYVSHLPVHQ DGSCNGLQHY AALGRDSVGA ASVNLEPSDV PQDVYSGVAA QVEVFRRQDA QRGMRVAQVL EGFITRKVVK QTVMTVVYG VTRYGGRLQI EKRLRELSDF PQEFVWEASH YLVRQVFKSL QEMFSGTRAI QHWLTESARL ISHMGSVVEW V TPLGVPVI QPYRLDSKVK QIGGGIQSIT YTHNGDISRK PNTRKQKNGF PPNFIHSLDS SHMMLTALHC YRKGLTFVSV HD CYWTHAA DVSVMNQVCR EQFVRLHSEP ILQDLSRFLV KRFCSEPQKI LEASQLKETL QAVPKPGAFD LEQVKRSTYF FS

UniProtKB: DNA-directed RNA polymerase, mitochondrial

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

MacromoleculeName: Non-Template Strand DNA (NT27mt_+1T) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.474738 KDa
SequenceString:
(DG)(DG)(DA)(DC)(DA)(DT)(DG)(DG)(DT)(DG) (DT)(DA)(DA)(DT)(DT)(DA)(DT)(DT)(DT)(DC) (DG)(DT)(DC)(DG)(DC)(DC)(DA)(DG)(DA) (DC)(DG)(DA)(DC)(DC)

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Macromolecule #5: Template Strand DNA (TS31mt_+1A)

MacromoleculeName: Template Strand DNA (TS31mt_+1A) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.428664 KDa
SequenceString:
(DG)(DG)(DT)(DC)(DG)(DT)(DC)(DT)(DG)(DG) (DC)(DG)(DA)(DG)(DC)(DG)(DC)(DG)(DC)(DC) (DG)(DT)(DT)(DA)(DC)(DA)(DC)(DC)(DA) (DT)(DG)(DT)(DC)(DC)

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Macromolecule #4: RNA (RNA14mt)

MacromoleculeName: RNA (RNA14mt) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.493723 KDa
SequenceString:
AGUCUGCGGC GCGC

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Macromolecule #6: DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER

MacromoleculeName: DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / type: ligand / ID: 6 / Number of copies: 1 / Formula: APC
Molecular weightTheoretical: 505.208 Da
Chemical component information

ChemComp-APC:
DIPHOSPHOMETHYLPHOSPHONIC ACID ADENOSYL ESTER / AMP-CPP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.9
Details: 20 mM Tris Buffer, pH 7.9, 150 mM NaCl, 10 mM DTT, and 5 mM MgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA Discharge
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details5 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
DetailsPreliminary grid screening performed manually using TFS Glacios.
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 21764 / Average exposure time: 1.89 sec. / Average electron dose: 50.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7998459
Details: Automated particle picking (Blob picker, CryoSPARC) with manual inspection (Inspect Particle Picks).
Startup modelType of model: OTHER / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF
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.
Number images used: 497782
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 2
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5ola


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial Fitting was done in Chimera and flexible/refined fitting done with Coot Phenix Real-Space Refinement.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 45 / Target criteria: Cross-correlation Coefficient
Output model

PDB-9bdd:
Cryo-EM Structure of Non-Cognate Substrate Bound in the Entry Site (ES) of Human Mitochondrial Transcription Elongation Complex

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