Significant changes between the X-ray structure and NMR structure of δ-subunit of RNA-polymerase

 

Gabriel Demo1,2, Veronika Papouskova1,2, Hana Sanderova4,5, Lukas Zidek1,2  and  Michaela Wimmerova1,2,3

 

1National Center for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic

2Central European Institute of Technology, Masaryk University, Brno, Czech Republic

3Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
4
Laboratory of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic,  Prague, Czech Republic

5Department of Bacteriology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic

guliver@mail.muni.cz

 

Keywords: δ-subunit of RNA-polymerase, X-ray, NMR, structure, discrepancies

RNA polymerase (RNAP) is an essential enzyme that is responsible for transcription of DNA into RNA. It is a multi-subunit enzyme and its composition is well conserved throughout all bacterial species. Gram-positive bacteria in comparison to gram-negative bacteria contain two additional subunits that associate with RNAP: δ [1] which is the subject of this work, and ω1 [2]. The recombinant form of δ-subunit from Bacillus subtilis is a 173 aa long protein with the acidic pI of 3.6. It was shown to consist of two domains: the N-terminal domain displaying an ordered structure as determined by CD spectroscopy, and the C-terminal domain, which appeared flexible and unstructured. The N-terminal domain was shown to interact with RNAP [3]. The structure of N-terminal domain was determined by NMR. It consists mainly of three α-helixes and two short β-sheets, yet the N-terminal part remains unstructured. The cause of this flexibility is probably the His6-Tag attached at the N-terminus [4].     

The N-terminal domain of δ-subunit was conquested to high-throughput screening (sitting drop), where several crystallization conditions were found. Further optimization showed two conditions that are more favourable for the crystal growth and it´s quality. Vapour diffusion hanging drop and under oil crystallization techniques were used for the crystallization in the optimizing steps. Higher quality crystals were obtained after 7-9 days. An iodine compound was used as a co-crystalline substance to have a possibility to provide a Single Anomalous Dispersion (SAD) experiments. Diffraction data were collected at BESSY II Berlin, beamline MX-14.2. The collection of SAD and native data of δ-subunit was successful. Both data were collected on the same crystal with average size 500 µm x 350 µm. The resolution of the SAD data was 1.66 Å and the native data 1.8 Å. The data were processed by MOSFLM and the structure was solved using SAD data by determination of the positions of heavy atoms, in this case iodines from the iodine substance used in optimizing step of the crystallization procedure. The space group was determined as C222(1), which means orthorhombic centro-symmetric space group. For the native data, the structure determination was done using molecular replacement with the structure solved from SAD data. The α-helical part of the protein is in a good agreement with the NMR structure. However,the X-ray structure showed completely different behaviour (folding) inthe region corresponding to β-sheets present in the NMR structure The problematic N-terminal part with His6-Tag on the end is not observable due to high flexibility. In the X-ray structure Ni2+ ions were observable, which can be explained by usage of Ni-NTA column for purification of the N-terminal domain of δ-subunit. Further investigations were concerned on the Ni2+ ions as a possible main factor of a different folding of the protein in the crystal structure. This is a strong proof that some of the proteins can be differently structured in the solution as in the crystalline phase.

References

1.       S. Clark, R. Losick, J. Pero, Nature, 252, (1974), 21–24.

2.       J. D. Helmann, C. P. Moran, Washington, DC: ASM Press, (2002) 289–312.

3.       F. J. Lopez de Saro, A. Y. Woody, J. D. Helmann, J. Mol. Biol., 252, (1995), 189–202.

4.       V. Motácková, H. Sanderová, L. Zídek, J. Novácek, P. Padrta, A. Svenková, J. Korelusová, J. Jonák, L. Krásný, V.   Sklenár, Proteins, 78(7), (2010), 1807-10.

 

Acknowledgements.

This work is supported by the Grant by CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund and Czech Science Foundation (GD301/09/H004).