Structural and biochemical characterization of RNA polymerase core and UvrD complex: a key component in transcription coupled DNA repair

Ravishankar Ramachandran.

SASDJL4 – RNA polymerase core subunit in complex with ATP-dependent DNA helicase UvrD1

DNA-directed RNA polymerase subunit alpha
DNA-directed RNA polymerase subunit beta
DNA-directed RNA polymerase subunit beta'
DNA-directed RNA polymerase subunit omega
ATP-dependent DNA helicase UvrD1
MWexperimental 458 kDa
MWexpected 448 kDa
VPorod 898 nm3
log I(s) 1.78×101 1.78×100 1.78×10-1 1.78×10-2
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 small angle scattering data  s, nm-1
ln I(s)
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 Guinier plot ln 1.78×101 Rg: 7.5 nm 0 (7.5 nm)-2 s2
(sRg)2I(s)/I(0)
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 Kratky plot 1.104 0 3 sRg
p(r)
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 pair distance distribution function Rg: 7.5 nm 0 Dmax: 22.2 nm

Data validation


Fits and models


log I(s)
 s, nm-1
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 DAMMIF model
DNA-directed RNA polymerase subunit alpha DNA-directed RNA polymerase subunit beta DNA-directed RNA polymerase subunit beta' DNA-directed RNA polymerase subunit omega ATP-dependent DNA helicase UvrD1 DAMMIN model

Synchrotron SAXS data from solutions of the RNA polymerase core subunit in complex with ATP-dependent DNA helicase UvrD1 in 10 mM Tris-Cl, 100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 5% Glycerol, pH 8 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.0992 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 6.40 mg/ml was measured at 4°C. 10 successive 1 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Sample is likely aggregated.

DNA-directed RNA polymerase subunit alpha
Mol. type   Protein
Organism   Mycobacterium tuberculosis
Olig. state   Dimer
Mon. MW   37.7 kDa
 
UniProt   P9WGZ1 (1-347)
Sequence   FASTA
 
DNA-directed RNA polymerase subunit beta
Mol. type   Protein
Organism   Mycobacterium tuberculosis
Olig. state   Monomer
Mon. MW   129.2 kDa
 
UniProt   P9WGY9 (7-1178)
Sequence   FASTA
 
DNA-directed RNA polymerase subunit beta'
Mol. type   Protein
Organism   Mycobacterium tuberculosis
Olig. state   Monomer
Mon. MW   146.7 kDa
 
UniProt   P9WGY7 (2-1316)
Sequence   FASTA
 
DNA-directed RNA polymerase subunit omega
Mol. type   Protein
Organism   Mycobacterium tuberculosis
Olig. state   Monomer
Mon. MW   11.8 kDa
 
UniProt   P9WGY5 (1-110)
Sequence   FASTA
 
ATP-dependent DNA helicase UvrD1
Mol. type   Protein
Organism   Mycobacterium tuberculosis
Olig. state   Monomer
Mon. MW   85.0 kDa
 
UniProt   P9WMQ1 (1-771)
Sequence   FASTA