Structural analysis of delta-subunit of RNA-polymerase by X-ray

 

Demo G.1, Papoušková V.1, Šanderová H.2,3, Wimmerová M.1

 

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

2Laboratory of Molecular Genetics of Bacteria, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

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

 

guliver@mail.muni.cz

 

RNA polymerase (RNAP) is an essential enzyme that is responsible for transcription of DNA into RNA. It is a multisubunit 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 173 aa long protein with an 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-helices and one short β-sheet, yet the N-terminal part remains unstructured. The cause of this flexibility is probably the His6-Tag attached at the N-terminus [4].

Therefore crystallization and structure determination by X-ray analysis were chosen. 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 its quality. Hanging drop was the technique used for the crystallization in the optimizing step. Crystals were obtained after 7-9 days with an average size 500 μm x 400 μm. Diffraction data were collected at ESRF Grenoble, ID-23-1. The data were processed by MOSFLM [5] and the determination of the structure was done by molecular replacement with NMR structure used as a model.

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

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

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

4.             V. Motáčková , H. Šanderová, L. Židek, J. Nováček, P. Padrta, A. Svenková, J. Korelusová, J. Jonák, L. Krásný, V. Sklenář, Proteins, 78(7), (2010), 1807-10

5.             A.G.W. Leslie, Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, 26, (1992)