Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

5A, 6A, 18B, NW12/2004G353

Purification, crystallization and preliminary X-ray diffraction analysis ofMethanococcus jannaschii TATA box-binding protein (TBP)
3
Naruhiko ADACHI1,2,3, Ryo NATSUME, Miki SENDA3, Shinsuke
MUTO1,2,
4
1,2Toshiya SENDA , Masami HORIKOSHI*
1Lab.
ofDev.
Biol.,IMCB,The Univ. of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
2
Horikoshi Gene Selector
Project,ERATO, JST, Tsukuba, Ibaraki 300-2635, Japan
3
JBIRC,JBIC,
Koto-ku,Tokyo 135-0064, Japan
4BIRC, AIST, Koto-ku, Tokyo 135-0064, Japan

Introduction
TATA box-binding protein (TBP) plays a central rolein gene expression through the interactions with RNA
polymerase and gene-specific transcription factors. As
TBP is one of the common components of eukaryotic andarchaeal transcription initiation, the investigation of thestructural and functional relationship of TBP in moredetail is the most attractive strategy to understand theunity and diversity of the mechanism of transcription
initiation among eukaryotes and archaea.
The tertiary structure of the core region of eukaryotic
TBP showed that TBP has a highly symmetric α/βstructure (βαβ4α)2 which contains a unique DNA binding
fold. That of archaeal TBP revealed that the folds ofarchaeal TBP are almost the same as those of eukaryotic
TBP, while that surface properties are quite different fromeukaryotic ones. The surface charge of eukaryotic TBP isstrongly basic whereas archaeal TBP are comparatively
neutral, implying that archaeal TBP have distinct
interacting factors from eukaryotic TBP.
Archaeal TBP can be divided into two groups as groupI and II, and only the members of group I are structurally
known [1]. Although the tertiary structure of group IIarchaeal TBP has not been solved, it is likely that group IIarchaeal TBP have different surface properties andinteracting factors from group I archaeal TBP. In fact, oneof the group II archaeal TBP (M. jannaschii TBP) showsa weak TATA box-binding activity, unlike eukaryotic andgroup I archaeal TBP. To analyze the conservation anddiversification of the mechanism of actions of TBP at theatomic level, we have initiated structural and functional
studies of M. jannaschii TBP. Here we report thepurification,crystallization
andpreliminary
crystallographic analysis of M. jannaschii TBP.
Methods
M. jannaschii TBP was purified as described earlier [1].
Crystals were grown within one month in three conditions
containing PEG 400, PEG 4000, and PEG MME 2000.
Further screenings for finding optimal conditions forcrystal growth were accomplished by varying the pH,
precipitant concentration, and volume of solution. Finally,
the best large crystals were obtained at 293K in a drop-1
containing 1.5 µl of 20 mg ml protein solution (20 mM
Tris-HCl (pH 7.9 at 277K), 10 % glycerol, 100 mM NaCl,
10 mM 2-mercaptoethanol), and 1.5 µl of reservoirsolution (0.2 M ammonium sulfate, 0.1 M sodium acetate
buffer pH 4.6, and 30 % PEG MME 2000). The data werecollected at 100 K from a single crystal using the ADSC
Quantum 315 CCD detector on the beamlines BL-5 at thePhoton Factory, Tsukuba, Japan. The diffraction data
were processed and scaled using the programs MOSFLMand SCALA, respectively.
Results
Crystals grew to approximate dimensions of 0.2 x 0.1 x
30.03 mm around one month. Examination of diffraction
data from M. jannaschii TBP crystals revealed that thesecrystals diffract beyond to 1.9 Å resolution and belong to
the space group P21, with unit-cell parameters a = 53.2 Å,
b = 55.5 Å, c = 123.4 Å, β = 91.0°. A value for theMatthews coefficient of 2.1 and a solvent content of
40.3 % were obtained assuming four molecules in theasymmetric unit. It was confirmed by a self-rotation map
which was calculated with the program POLARRFN in
the CCP4 program suite. The self-rotation map suggeststhat there are two non-crystallographic two-fold axes.
Molecular replacement (MR) was performed with theprogram MOLREP. Further crystallographic refinement isin progress.
Table 1: Data-collection and processing statistics
Space group
P21
Unit-cell parameters
a = 53.2, b = 55.5,
c = 123.4 Å,
β = 91.0°
Resolution range
62.00 – 1.90
No. of measured reflections
208543
No. of unique reflections
57094
Rmerge # (%)
3.9 (35.0)$
Completeness (%)
99.9 (99.9)$$
9.2 (2.1)
Average I/σ#Rmerge = Σ|I(h)-<I(h)>|/ΣI(h)$Last shell, 2.00 – 1.90 Å.
References[1] N. Adachi et al., Acta. Crystallogr. D 60, 2328 (2004).
* horikosh@iam.u-tokyo.ac.jp

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