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
4Laboratory
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.
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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).