Exploring RNA polymerase regulation by NMR spectroscopy

RNA synthesis is a central process in all organisms, with RNA polymerase (RNAP) as the key enzyme. Multisubunit RNAPs are evolutionary related and are tightly regulated by a multitude of transcription factors. Although Escherichia coli RNAP has been studied extensively, only little information is available about its dynamics and transient interactions. This information, however, are crucial for the complete understanding of transcription regulation in atomic detail. To study RNAP by NMR spectroscopy we developed a highly efficient procedure for the assembly of active RNAP from separately expressed subunits that allows specific labeling of the individual constituents. We recorded [1H,13C] correlation spectra of isoleucine, leucine, and valine methyl groups of complete RNAP and the separately labeled β’ subunit within reconstituted RNAP. We further produced all RNAP subunits individually, established experiments to determine which RNAP subunit a certain regulator binds to, and identified the β subunit to bind NusE.


Supplementary Methods
Cloning. Plasmids containing the genes rpoA, rpoB, rpoC and rpoZ were kindly provided by Irina Artsimovitch. rpoB was cloned from pIA942 into pET29b (Novagen, Madison, WI, USA) via BamHI and NdeI. rpoC was cloned from pIA661 into pET29b via NdeI and HindIII restriction sites allowing the expression of rpoC with a hexahistidine tag at the C-terminus. For tagless production of rpoZ the gene was excised from pIA839 with its ribosome binding site via XbaI and HindIII and rpoC was expressed in E. coli Rosetta (DE3) pLysS (Novagen, Madison, WI, USA). The recombinant protein harbored a seven amino acid linker followed by a hexahistidine tag (His 6 ) at the C-terminus. Cells were grown in M9 minimal medium 3,4 containing 30 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C. When an OD 600 of ~ 0.5 was reached the temperature was lowered to 16 °C and gene expression was induced with 1 mM IPTG at an OD 600 of 0.6-0.8. Cells were harvested 6 h after induction, resuspended and lysed as described above using buffer A (50 mM Tris/HCl (pH 7.5), 500 mM NaCl, 5 % (v/v) glycerol, 10 mM MgCl 2 , 10 µM ZnCl 2 , 10 mM imidazole). After centrifugation (30 min, 12,000 x g, 4 °C) the supernatant was applied to a HisTrap HP column (GE Healthcare, Munich, Germany). After washing with buffer A, elution was carried out using a step gradient with increasing imidazole concentrations (10-500 mM in buffer A).
Fractions containing β' were combined. Following dialysis against the required buffer the protein solution was concentrated by ultrafiltration (MWCO = 10 kDa) and stored at -80 °C after shock freezing in liquid nitrogen. One liter culture yielded 15 mg protein.
The ω subunit with N-terminal His 6 -SUMO tag was produced in E. coli Rosetta (DE3) pLysS harboring pET28M-SUMO1/rpoZ. Cells were grown in M9 minimal medium 3,4 in the presence of 30 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C until an OD 600 of 0.4 was reached.
The temperature was lowered to 25 °C and at an OD 600 of 0.6-0.8 expression was induced with 1 mM IPTG. Cells were harvested after 4 h, resuspended and lysed as described above. In this case 25 mM Tris/HCl (pH 7.5), 300 mM NaCl, 10 mM imidazole was used for resuspension. After centrifugation (12,000 x g, 30 min, 4 °C), the supernatant was applied to a HisTrap HP column.
After washing with 25 mM Tris/HCl (pH 7.5), 300 mM NaCl, 10 mM imidazole, elution was performed using a step gradient with increasing imidazole concentrations (10-500 mM in resuspension buffer). Fractions containing His 6 -SUMO-ω were combined and cleaved during dialysis overnight against 25 mM Tris/HCl (pH 7.5), 300 mM NaCl by Senp2, a protease that cleaves directly after SUMO protein. The protein solution was reapplied to the HisTrap HP column.
Pure ω was found in the flow through, dialyzed against the required buffer, concentrated by ultrafiltration (MWCO = 3 kDa) and stored at -80 °C after freezing with liquid nitrogen with a yield of 3 mg protein per liter culture.

NusA-NTD
Formaldehyde crosslink. The crosslinking of RNAP and NusB:NusE Δ was based on the SPINE method 10 . 7.7 nmol RNAP were mixed with 15.4 nmol NusB:NusE ⊗ in 25 mM HEPES (pH 7.5), 100 mM NaCl and a 4 % (w/v) paraformaldehyde solution in the same buffer was added to a final concentration of 0.6 % (w/v). For the crosslink, the mixture was incubated at 37 °C for 20 min. 0.7 ml of Ni 2+ chelating sepharose (50 % (w/v), GE Healthcare, Munich, Germany), equilibrated with 25 mM HEPES (pH 7.5), 100 mM NaCl, were added and incubated for 20 min at room temperature. Afterwards the mixture was transferred to a 2.5 ml gravity flow column and the flow trough was collected. The column was washed ten times with 1 ml of 25 mM HEPES (pH 7.5),