The C-terminal WRKY domain of Arabidopsis thaliana WRKY1 protein, a transcription factor, was cloned and expressed. The expressed protein was then purified and crystallized. The preliminary X-ray analysis was undertaken. The crystal diffracted to 2.50 A resolution in-house and belongs to space group P2(1) with unit-cell parameters a=64.10 A, b=34.88 A, c=114.72 A, beta=90.49 degrees .
"The pellet was washed with PBS, resuspended and sonicated in buffer A (20 mM Tris–HCl, pH 7.5, 1 M NaCl). P8 was purified using a pre-packed 1 ml Hi-Trap (Pharmacia) Ni column as described by Duan et al. , and further desalted against buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl). "
[Show abstract][Hide abstract] ABSTRACT: Virus-encoding nuclear transcriptional regulators play important roles in the viral life cycle. Most of these proteins exhibit intrinsic transcriptional activation or repression activity, and are involved in the regulation of the expression of virus genome itself or important cellular genes to facilitate viral replication and inhibit antiviral responses. Here, we report that the minor core protein P8 of Rice black-streaked dwarf virus, a dsRNA virus infecting host plants and insects, is targeted to the nucleus of insect and plant cells via its N-terminal 1-40 amino acids and possesses potent active transcriptional repression activity in Bright Yellow-2 tobacco suspension cells. Moreover, P8, like many transcriptional regulatory proteins, is capable of forming homo-dimers within insect cells and in vitro. All these data suggest that P8 is likely to enter the nucleus of host cell and play an important role as a negative transcriptional regulator of host gene expression during the process of virus-host interaction.
[Show abstract][Hide abstract] ABSTRACT: A large-scale, high-efficiency and low-cost platform based on a Beckman Coulter Biomek FX and custom-made automation systems for structural genomics has been set up at Peking University, Beijing, People's Republic of China. This platform has the capacity to process up to 2000 genes per year for structural and functional analyses. Bacillus subtilis, a model organism for Gram-positive bacteria, and Streptococcus mutans, a major pathogen of dental caries, were selected as the main targets. To date, more than 470 B. subtilis and 1200 S. mutans proteins and hundreds of proteins from other sources, including human liver proteins, have been selected as targets for this platform. The selected genes are mainly related to important metabolism pathways and/or have potential relevance for drug design. To date, 40 independent structures have been determined; of these 11 are in the category of novel structures by the criterion of having less than 30% sequence identity to known structures. More than 13 structures were determined by SAD/MAD phasing. The macromolecular crystallography beamline at the Beijing Synchrotron Radiation Facility and modern phasing programs have been crucial components of the operation of the platform. The idea and practice of the genomic approach have been successfully adopted in a moderately funded structural biology program and it is believed this adaptation will greatly improve the production of protein structures. The goal is to be able to solve a protein structure of moderate difficulty at a cost about US 10,000 dollars.
[Show abstract][Hide abstract] ABSTRACT: WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
Nucleic Acids Research 02/2007; 35(4):1145-54. DOI:10.1093/nar/gkm001 · 9.11 Impact Factor
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