Structure determination of an RNA polymerase subunit unique for gram-positive bacteria
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| Year of publication | 2010 |
| Type | Conference abstract |
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| Description | RNA polymerase (RNAP) is an essential multisubunit enzyme responsible for transcription of genetic information from DNA into RNA. The architecture of the RNA polymerase from Bacillus subtilis and other gram-positive bacteria differs from its analogue from gram-negative bacteria in the presence of two additional subunits - omega1 and delta. Their role in the transcription machinery is still not well understood. It has been proposed that the delta subunit is important for the virulence of Staphylococcus aureus and Streptococcus agalactiae which makes it interesting also from the medical point of view. Recent results of our collaborators (Libor Krásný et al., Institute of Microbiology of the Academy of Sciences of the Czech Republic) indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. Therefore, we focused on the delta subunit in our structural study. Because a 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. This domain is proposed to bind to the RNAP core and probably orients the C-terminal domain on the surface of RNAP. Moreover, the C-terminal domain is highly negatively charged which might mimic nucleic acids and compete with DNA/RNA for binding on RNAP. The N-terminal delta domain was prepared using a standard protocol of overexpression in the E. coli system to produce a 15N,13C-uniformly labeled sample. A basic set of triple resonance NMR experiments was measured and all resonances of the backbone nuclei were assigned. Resonance frequencies of the side-chains were assigned using 3D TOCSY- and NOESY-type spectra. Secondary structure was predicted based on medium-range NOEs, three-bond HN-HA couplings, and chemical shifts analyzed by programs CSI and TALOS. 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 in the SCULPTOR CNS module. Programs CING, PROCHECK, and WHATIF were used to check the quality of the calculated structures. The chemical shifts and calculated structures have been deposited in the BMRB and PDB databases, respectively. The determined structure allowed us to identify unexpected structure homology of the N-terminal domain of RNAP delta subunit with some proteins from the Forkhead DNA/RNA-binding domain SCOP family. Results of this study have been accepted for publication (Motáčková et al.: Solution structure of the N-terminal domain of Bacillus subtilis delta subunit of RNA polymerase and its classification based on structural homologs, Proteins, 2010, in press). |
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