SPring-8, the large synchrotron radiation facility

Life Science

Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution

The DNA-dependent RNA polymerase (RNAP) is the key enzyme of the transcription process, and is a final target in many regulatory pathways that control gene expression in all living organisms. In bacteria, RNAP is responsible for the synthesis of all RNAs in the cell (mRNA, rRNA, tRNA, etc.). The transcription cycle could be roughly divided into three major steps: initiation, elongation, and termination. The bacterial RNAP exists in two forms: core and holoenzyme. The core enzyme has a molecular mass of ~400 kDa, and consists of five subunits: α-dimer, β, β', and ω. Although it is active in elongation, the core enzyme is incapable of initiating transcription efficiently and with specificity. For this, it must bind an initiation factor, σ, to form a holoenzyme that can recognize specific DNA sequences (promoters).
Though the RNAP core enzymes are evolutionarily conserved in sequence, structure, and function from bacteria to human, the bacterial and eukaryotic initiation factors, which are required to form the holoenzymes, reveal no similarity. Thus, the bacterial RNAP holoenzyme is an important and promising target for design of effective antibiotics against the pathogenic bacteria (like Mycobacterium tuberculosis).
We have determined the crystal structure of the T. thermophilus RNAP holoenzyme (molecular mass ~450 kDa), containing the major σ-factor (σ70), at 2.6 Å resolution using the RIKEN beamline (BL45XU) for data collection. The structure provides insight into the different stages of the bacterial transcription initiation at the atomic level and can be effectively used in drug design to develop novel bacteria-specific inhibitors of transcription.

provided by Shigeyuki Yokoyama, RIKEN/University of Tokyo

Holoenzyme crystal structure. The subunits colors are:β, sage; β', white (β'163-452, cyan; β' Zn-finger, green); αI, blue; αII, light orange; σ, magenta; and ω, red. Two catalytic Mg2+ (red) and two Zn2+ ions (blue) are shown as spheres.

Insight into the mechanism of active transport by calcium pump

SPring-8 played a vital role in the recent structure determinations of the sarcoplasmic reticulum (SR) calcium pump in the calcium bound and unbound states. The ATP-driven calcium pump is an integral membrane protein (molecular weight of 110k) that relaxes muscle cells by pumping calcium released during contraction back into the sarcoplasmic reticulum. The crystals were thin (<20 µm; Ca²⁺-bound form) or had a very large unit cell dimension (nearly 600 Å; Ca²⁺-unbound form). Hence, the use of very bright and highly parallel X-ray beam available in undulator beamlines, such as BL41XU (Structural biology I) and BL44XU (Protein Institute, Osaka University), were essential to these structure determinations.
These studies have revealed that the binding of calcium alone accompanies a surprisingly large-scale rearrangement of both transmembrane and cytoplasmic domains. and that the ion pumps work like mechanical pumps at an atomic scale. Also, the structure of a very strong inhibitor, thapsigargin (TG), bound to this pump was determined and may serve as a template for drugs targeted for membrane proteins. Calcium is a fundamental and ubiquitous factor in the regulation of intracellular processes. Therefore, the atomic structures of the calcium pump in different states have a tremendous impact on many fields, including medical treatment for myocardial diseases and cancer.

provided by Chikashi Toyoshima, University of Tokyo

Structural changes in calcium pump

X-ray diffraction pattern recorded from a single myofibril of muscle

A striated muscle consists of many muscle cells, in which filaments of contractile proteins are arranged in regular hexagonal lattices. Because of this arrangement, a muscle gives rise to a series of reflections when irradiated with X-rays. The smallest muscle specimen used for X-ray diffraction studies has been a single muscle cell (diameter, 50 - 100 µm). By using the BL45XU, diffraction patterns were record-ed for the first time from a single myofibril, the smallest building block of a muscle cell (diameter, only 1 - 3 µm). A single myofibril contains only one hexagonal lattice of protein filaments.
Figure 1 shows a diffraction pattern recorded from a single myofibril of bumblebee flight muscle. It is clear from the hexagonally symmetrical arrangement of diffraction spots that the pattern comes from a single hexagonal lattice. The recording was made possible by the combination of the use of X-ray microbeams (diameter, 2 µm) and the end-on irradiation technique, in which the beam path was made parallel to the myofibrillar axis (Fig. 2). The specimen used in Fig. 1 was ~3 mm long, and the spot-like reflections imply that there was not even a slightest twist of the hexagonal lattice along the entire length of the myofibril. Such an extraordinary register of protein filaments (each filament is only ~2 µm in length) may be specific to the flight muscle of higher insects capable of sophisticated flight maneuvers.

provided by Hiroyuki Iwamoto, JASRI

Fig. 1		Fig. 2

Switch mechanism for polymorphic supercoiling of the bacterial flagellar filament

The bacterial flagellar filament is a helical propeller. A rotary motor at its base drives the rotation of the filament at around 300 Hz. Its high-speed rotation drives the swimming of the cell, and the switching of its helical hand by quick reversal of the motor causes the tumbling for chemotaxis and thermotaxis. The filament is a tubular structure made by helical assembly of a single protein, flagellin, and it is also made of 11 protofilaments. Precisely determined packing arrangements of flagellin subunits in two slightly different conformations produce various but well defined forms of supercoils with different helical hand and pitch. Previous studies by electron cryomicroscopy revealed the molecular shape, domain organization and packing arrangements of flagellin in the filament. Now, X-ray crystallographic analysis of the flagellin core fragment from multiple anomalous diffraction data sets collected at BL45XU revealed the protofilament structure and the molecular interactions responsible for the axial assembly of flagellin in atomic detail.
Since the flagellar protofilament switches between the two conformations with axial repeats of 51.9 Å and 52.7 Å to produce gentle curvatures of flagellar supercoils, and the protofilament structure obtained was the one with the shorter repeat, a computational simulation of an extension of the protofilament model was carried out to identify the conformational switch in the molecular structure of flagellin. After gradual axial elongation of the molecular structure by about a few Å, it showed a subtle but significant jump in the conformation of a β-hairpin located at the molecular interface that forms the protofil-ament. This demonstrated the characteristic nature of proteins that perform flexible and yet highly precise functions as nanomachine.

provided by Keiichi Nanba, JASRI/Osaka University