Veronika Motáčková


National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlářská 2, 61137 Brno, Czech Republic


RNA polymerase is an essential multisubunit enzyme responsible for transcription of genetic information from DNA into RNA. The architecture of the RNA polymerase from Bacillus subtilis differs from its analogue from gram-negative bacteria in a presence of two additional subunits – omega1 and delta. Their role in the transcription machinery is still not fully clear. Recent results of our collaborators (L.Krásný et al., Inst. of Microbiology, ASCR, Prague) indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. Some reports also showed the importance of the delta subunit for the virulence of Staphylococcus aureus and Streptococcus agalactiae. Therefore, we focused on the delta subunit to reveal its structure and related dynamics. Because the C-terminal domain of the delta subunit is unstructured and its sequence is highly repetitive, we started a systematic investigation of the protein with a shorten construct, corresponding to the well-structured N-terminal part. 

The N-terminal domain of the delta protein was prepared using a standard protocol of overexpression in the E.coli system to produce a 15N,13C-uniformly labeled sample. All spectra, including a standard set of triple resonance NMR experiments, 3D TOCSY, and 3D NOESY, were measured on a 600MHz spectrometer. The secondary structure was predicted based on the chemical shifts of backbone nuclei in program TALOS, three-bond scalar couplings, and medium range NOEs. The distance restrains were extracted and assigned from NOESY spectra using program ARIA 2.1. The additional RDC restraints from two aligning media (bacteriophage Pf1, 5% polyacrylamide gel) and anisotropic contributions to the 13C chemical shifts were used in the final refinement, employing the SCULPTOR CNS module. The quality of the calculated structures were checked by programs CING, PROCHECK, and WHATIF. The determined structure allowed us to identify unexpected structure homology with some proteins from the “Forkhead DNA-binding domain” SCOP family.

Fast ns-ps backbone motions of the N-terminal domain of delta subunit was observed using the standard set of  15N relaxation experiments (R1, R2, NOE) performed at two magnetic field (500MHz, 600MHz). The relaxation experiments were acquired at two temperatures (300K, 280K). Slow ms-us motions were investigated by CPMG and T1rho relaxation dispersion experiments.


This work was supported by the Grants MSM0021622413 and LC06030 from the Ministry of Education, Youth and Physical Culture of the Czech Republic, by the Grant 204/09/0583 from Czech Science Foundation, and FRVŠ 1851/2010.