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- PDB-8abz: RNA polymerase at U-rich pause bound to non-regulatory RNA - paus... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8abz | |||||||||||||||
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Title | RNA polymerase at U-rich pause bound to non-regulatory RNA - pause prone, closed clamp state | |||||||||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||||||||
Method | ![]() ![]() ![]() | |||||||||||||||
![]() | Dey, S. / Weixlbaumer, A. | |||||||||||||||
Funding support | European Union, ![]()
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![]() | ![]() Title: Structural insights into RNA-mediated transcription regulation in bacteria. Authors: Sanjay Dey / Claire Batisse / Jinal Shukla / Michael W Webster / Maria Takacs / Charlotte Saint-André / Albert Weixlbaumer / ![]() Abstract: RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at ...RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination. | |||||||||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 606.7 KB | Display | ![]() |
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PDB format | ![]() | 469.5 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 15328MC ![]() 8abyC ![]() 8ac0C ![]() 8ac1C ![]() 8ac2C ![]() 8acpC ![]() 8ad1C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: Protein | ![]() Mass: 36558.680 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() #2: Protein | | ![]() Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950 Production host: ![]() ![]() ![]() ![]() #3: Protein | | ![]() Mass: 155237.672 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() Gene: rpoC, A6592_25550, A9X72_23915, AAG43_004319, ABE90_012715, ACN68_05320, ACN81_23175, ACU57_23185, AM464_13270, APX88_03320, AT335_003682, AT845_003984, AW119_26060, B6R12_002490, B6R15_000400, ...Gene: rpoC, A6592_25550, A9X72_23915, AAG43_004319, ABE90_012715, ACN68_05320, ACN81_23175, ACU57_23185, AM464_13270, APX88_03320, AT335_003682, AT845_003984, AW119_26060, B6R12_002490, B6R15_000400, B6R31_004826, B6R48_004921, B6R87_004892, BANRA_00556, BANRA_02658, BANRA_05060, BEA19_22935, BF481_004768, BG944_004752, BGZ_01811, BGZ_04901, BHS81_23830, BJI68_24535, BJJ90_25120, BK292_28025, BK383_27720, BMC79_004282, BMT49_21005, BMT50_08090, BMT91_03285, BN17_39521, BO068_004210, BOH76_00535, BON63_20110, BON64_17760, BON65_15085, BON66_14380, BON67_10880, BON68_25620, BON69_07675, BON70_00395, BON71_08940, BON72_17275, BON73_00245, BON74_15225, BON75_02240, BON76_16220, BON77_22675, BON78_03480, BON79_17585, BON80_22860, BON81_01110, BON83_01620, BON87_25670, BON89_14770, BON91_27070, BON92_00590, BON93_00705, BON94_22185, BON95_12860, BON96_11000, BON97_20250, BON98_22915, BTQ06_27265, BvCmsF30A_00478, BvCmsHHP019_04451, BvCmsHHP056_02607, BvCmsKKP061_02982, BvCmsKSNP073_02854, BvCmsNSP072_02893, BVL39_05360, BXT93_12730, BZL69_05725, C2U48_12715, C5N07_24540, C5Y87_25310, C9114_22905, C9160_23380, C9E67_28655, CA593_06020, CCS08_08140, CDC27_21560, CDL36_24870, CG831_003924, CIG67_06970, CO706_19510, CR538_24210, CR539_01675, CV83915_02065, CWS33_24550, CXJ73_004162, CY655_25300, D0X26_26125, D3822_07080, D3Y67_18045, D9D43_22315, D9D77_23620, D9E34_21770, D9H94_18950, D9J03_21580, DAH17_22710, DAH18_24925, DAH19_24515, DAH20_24815, DAH21_23320, DAH23_25900, DAH24_26040, DAH25_26075, DAH26_25940, DAH27_25270, DAH28_23885, DAH29_24350, DAH30_23845, DAH31_24375, DAH33_24565, DAH34_21555, DAH35_26340, DAH36_23725, DAH37_22570, DAH38_23230, DAH40_23335, DAH42_24295, DAH43_24880, DAH45_24825, DAH46_24660, DAH47_25135, DAH48_24925, DAH49_24835, DAH50_22035, DD762_22280, DEN87_24580, DEN88_24360, DEN89_26510, DEN90_25535, DEN91_25230, DEN92_24665, DEN93_24980, DEN94_24465, DEN95_25125, DEN96_23600, DEN97_23710, DEN98_23795, DEN99_23135, DEO00_23170, DEO01_23750, DEO02_23980, DEO03_22675, DEO04_24305, DEO05_24175, DEO06_24620, DEO07_24855, DEO08_24290, DEO09_24370, DEO10_24290, DEO11_23940, DEO12_25050, DEO13_24310, DEO14_24495, DEO15_24970, DEO17_24875, DEO18_23920, DEO19_24555, DEO20_25020, DIV22_03745, DN627_23155, DRW19_23990, DTL43_13700, DTL90_24530, DTM16_24360, DU321_23425, DXT69_22805, DXT70_21465, DXT71_22310, DXT73_22940, E0I42_21900, E2112_20360, E2113_19800, E2117_21880, E2119_22340, E2121_20400, E2122_20800, E2127_20900, E2128_03185, E2129_21295, E2131_19275, E2132_20135, E2133_18885, E2134_22790, E2135_19330, E3O05_23740, E4K51_22920, E4T14_19265, E5M02_21775, E5P22_12650, E5P23_19725, E5P24_19615, E5P25_23055, E5P26_18290, E5P27_19155, E5P28_08825, E5P29_14400, E5P30_23670, E5P31_18530, E5P32_02825, E5P33_07130, E5P34_19120, E5P35_02265, E5P36_06040, E5P37_21450, E5P39_19425, E5P40_03200, E5P41_16050, E5P42_04855, E5P43_16470, E5P44_18160, E5P45_17320, E5P46_15010, E5P47_19430, E5P48_14465, E5P49_19235, E5P50_22485, E5P51_03205, E5P52_19925, E5S34_21385, E5S35_21950, E5S36_22335, E5S38_21405, E5S39_21135, E5S42_21540, E5S43_20990, E5S44_22255, E5S45_22685, E5S47_23025, E5S48_22965, E5S51_21950, E5S52_22260, E5S53_21960, E5S54_23760, E5S55_21995, E5S56_22725, E5S57_22160, E5S58_21685, E5S61_22375, E5S62_23450, EAI46_11695, EAN77_22650, EAX79_24760, EBP16_21385, EC1094V2_4282, ECs4911, ED648_18905, EHD79_23525, EHH55_00715, EI021_22070, EIZ93_16335, EL79_4407, EL80_4314, ELT15_18455, ELT17_21010, ELT18_21900, ELT20_20720, ELT21_20545, ELT23_22660, ELT27_20990, ELT29_22075, ELT38_22790, ELT41_20490, ELT57_22290, ELU85_23590, ELU87_21975, ELU90_21605, ELU99_23055, ELV08_18690, ELV09_17955, ELV21_16085, ELV26_23220, ELV28_22250, ELX48_22415, ELX57_20105, ELX61_20545, ELX65_19750, ELX66_22520, ELX67_21035, ELX69_20060, ELX72_20720, ELX74_23585, ELX76_21250, ELX78_21735, ELX82_21250, ELX84_22555, ELX85_02100, ELX86_18600, ELX87_22155, ELX90_21365, ELX91_23125, ELX96_15665, ELX98_18555, ELY02_19945, ELY05_18165, ELY18_20340, ELY22_19955, ELY30_22450, ELY31_22565, ELY32_24365, ELY35_18500, ELY36_23180, ELY39_21195, ELY41_16445, ELY48_22270, ELY49_19025, ERS139208_04657, EVY14_16840, EYV17_20985, EYV18_01360, F0L67_26015, F2N31_22415, F3N40_21595, F7F11_23815, F9B07_23670, F9V24_17840, F9X20_05965, FDM60_19485, FFF58_24430, FJQ51_21480, FOI11_015710, FOI11_24350, FQF29_20795, FV293_23430, FVB16_05290, FWK02_01680, FY127_17750, G3813_004460, G4A38_22920, G5632_19555, GAJ26_24175, GF147_25310, GF699_23055, GFY34_22475, GIB53_18270, GJ11_25200, GKF89_20995, GNZ05_25260, GP662_20930, GP946_22700, GP954_05255, GP979_07400, GQA06_03470, GQE64_22340, GQF59_23130, GQM04_07520, GQM09_18365, GQM13_25045, GQW68_22045, GRW05_10055, GRW57_01390, GRW81_21775, GSY44_21840, GUB08_21175, GUC01_23005, H0O72_20910, HHH44_004417, HI055_004311, HJQ60_004916, HJS37_004852, HJU54_004824, HKA49_004956, HL563_20665, HLZ50_21730, HMU48_21580, HMV95_21230, HNC36_22380, HNC59_22525, HNC66_25345, HNC99_22045, HV109_22420, HV209_21190, HVW19_23415, HVW43_14980, HVX16_24445, HVX31_19105, HVZ30_19815, HVZ71_23865, HX136_23635, I6H00_17165, I6H01_14335, I6H02_15720, IH768_05165, IH772_15235, IT029_004865, J0541_004717, J4S20_004535, J5U05_003963, JE86ST02C_45250, JE86ST05C_45350, JFD_05094, JNP96_01280, NCTC10082_02080, NCTC10090_02779, NCTC10429_00081, NCTC10865_05894, NCTC10974_05350, NCTC11181_01964, NCTC12950_05213, NCTC13127_06163, NCTC13148_04414, NCTC13216_02108, NCTC7927_05248, NCTC8008_04280, NCTC8333_05557, NCTC8500_05324, NCTC8960_02339, NCTC9036_04648, NCTC9037_04781, NCTC9044_02305, NCTC9045_05541, NCTC9077_05932, NCTC9111_04954, NCTC9706_02002, NCTC9777_01117, ND22_004792, PGD_00622, RG28_25345, SAMEA3472044_04873, SAMEA3472056_05309, SAMEA3472067_05183, SAMEA3472080_04815, SAMEA3472147_04120, SAMEA3751407_05007, SAMEA3752557_04844, SAMEA3753106_04232, WP2S18E08_45640, WP4S18E07_42680, WR15_01490 Production host: ![]() ![]() ![]() ![]() #4: Protein | | ![]() Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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-DNA chain , 2 types, 2 molecules NT
#5: DNA chain | Mass: 91015.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
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#7: DNA chain | Mass: 91180.203 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
-RNA chain , 1 types, 1 molecules R
#6: RNA chain | Mass: 39226.262 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
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-Non-polymers , 2 types, 3 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/ZN.gif)
#8: Chemical | ChemComp-MG / |
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#9: Chemical |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Escherichia coli RNA polymerase non-regulatory RNA complex - pause prone, closed clamp state Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.435 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() ![]() |
Buffer solution | pH: 8 Details: 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT |
Specimen | Conc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() Details: The co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs ...Details: The co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs halted at the U-rich pause (G93). The complexes were prepared in 0.03 ml reactions and contained: 0.02 mM of E. coli RNAP, 0.080 mM of E. coli Sigma70, 0.02 mM of template DNA, 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT and 6 mM of each ATP, GTP, CTP and UTP. Initially, all the components except NTPs were added and incubated for 10 min at 310K. To allow transcription elongation, NTPs were added and the sample was incubated for 20 to 40 min at 310K. The final products were purified on a Superose 6 Increase 3.2/300 gel filtration column equilibrated in Glutamate buffer. Samples were concentrated to 10-12 mg/mL using an Amicon Ultra 0.5mL centrifugal filter unit (30 KDa MWCO). Before grid freezing, 8 mM of CHAPSO was added to freshly prepared sample to overcome preferred particle orientation. |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R1.2/1.3 |
Vitrification![]() | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture ...Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture prior to the application of 0.004 ml of sample. Grids were plunge frozen into liquid ethane using a Vitrobot mark IV (FEI) with 95% chamber humidity at 283K. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.2 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 |
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Processing
Software |
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EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180841 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: map cross correlation | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6ALH | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 51.34 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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