SASDFB8: Delta subunit of RNA polymerase, RNAP (B. subtilis): Lysine to gl...

Basic information

Entry

Database: SASBDB / ID: SASDFB8

Sample

Delta subunit of RNA polymerase, RNAP (B. subtilis): Lysine to glutamate mutant, 400mM NaCl

• DNA-directed RNA polymerase subunit delta - mutantPolymerase (protein), rpoE KE mutant, Bacillus subtilis (strain 168)

Function / homology

Function:

DNA-directed RNA polymerase complex / DNA-directed 5'-3' RNA polymerase activity / DNA-templated transcription / regulation of DNA-templated transcription

Domain/homology:

DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile.

Component:

DNA-directed RNA polymerase subunit delta

• Biological species: Bacillus subtilis (strain 168) (bacteria)
• Citation: Journal: J Am Chem Soc / Year: 2019; Title: Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase.; Authors: Vojtěch Kubáň / Pavel Srb / Hana Štégnerová / Petr Padrta / Milan Zachrdla / Zuzana Jaseňáková / Hana Šanderová / Dragana Vítovská / Libor Krásný / Tomáš Koval' / Jan Dohnálek / Joanna Ziemska-Legiecka / Marcin Grynberg / Patryk Jarnot / Aleksandra Gruca / Malene Ringkjøbing Jensen / Martin Blackledge / Lukáš Žídek / ; Abstract: Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.

Contact author

• Vojtěch Kubáň (MUNI, Masaryk University; Research group Structure and Dynamics of Proteins., Brno, Czech Republic)

Models

Sample

• Sample: Name: Delta subunit of RNA polymerase, RNAP (B. subtilis): Lysine to glutamate mutant, 400mM NaCl; Specimen concentration: 2.6 mg/ml
• Buffer: Name: 20 mM Phosphate buffer, 400 mM NaCl, 0.05% NaN3 / pH: 6.6

Entity #1738

Name: rpoE KE mutant / Type: protein
Description: DNA-directed RNA polymerase subunit delta - mutantPolymerase
Formula weight: 20.405 / Num. of mol.: 1 / Source: Bacillus subtilis (strain 168) / References: UniProt: P12464
Sequence:

MGIKQYSQEE LKEMALVEIA HELFEEHKKP VPFQELLNEI ASLLGVKKEE LGDRIAQFYT DLNIDGRFLA LSDQTWGLRS WYPYDQLDEE TQPTVEAEEE EAEEAVEEDL DLDEFEEIDE DDLDLDEVEE ELDLEADDFD EEDLDEDDDD LEIEEDIIDE DDEDYDDEEE EIK

Experimental information

• Beam: Instrument name: PETRA III EMBL P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.124 Å / Dist. spec. to detc.: 3 mm
• Detector: Name: Pilatus 2M

Scan

Title: Delta subunit of RNA polymerase, RNAP (B. subtilis): Lysine to glutamate mutant, 400mM NaCl
Measurement date: Oct 3, 2016 / Storage temperature: 20 °C / Cell temperature: 20 °C / Exposure time: 0.05 sec. / Number of frames: 20 / Unit: 1/nm /

	Min	Max
Q	0.0134	3.7925

Result

Type of curve: single_conc /

	Experimental	Porod
MW	28.9 kDa	49 kDa
Volume	-	78 nm3

	Guinier	Guinier error
Forward scattering, I0	4417	22
Radius of gyration, Rg	4.49 nm	0.82

	Min	Max
D	-	24
Guinier point	30	133