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Crystallographic characterization of Pap1-DNA complex
1Department of Molecular Biology, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-01, Japan.
Abstract:
Pap1 is a fission yeast transcription factor that activates genes related with resistance against staurosporine, a potent inhibitor of protein kinase C, and has been shown to be involved in cell growth, cell cycle, carcinogenesis and differentiation. Pap1 has the bZIP DNA-binding domain but binds to non-consensus DNA sequences for the bZIP motif. Highly ordered crystals of the DNA-binding domain complexed with a DNA fragment that has an ATF/CREB-like non-consensus sequence have been obtained. The crystals grew by the vapor-diffusion technique with polyethylene glycol 6000 and belong to space group R3 with a = b = 240.78, c = 43.85 A. A 2.0 A resolution data set was collected with a cryo-crystallographic technique.
Insights
Pap1, a fission yeast transcription factor, binds non-consensus DNA sequences. Researchers crystallized the Pap1 DNA-binding domain with a non-consensus DNA fragment to understand its function.
Area of Science:
- Molecular Biology
- Structural Biology
- Yeast Genetics
Background:
- Pap1 is a fission yeast transcription factor regulating genes involved in staurosporine resistance, cell growth, cell cycle, carcinogenesis, and differentiation.
- Pap1 possesses a basic leucine zipper (bZIP) DNA-binding domain but interacts with non-consensus DNA sequences, diverging from typical bZIP binding motifs.
Purpose of the Study:
- To elucidate the structural basis of Pap1's interaction with non-consensus DNA sequences.
- To obtain high-resolution structural data of the Pap1 DNA-binding domain complexed with its target DNA.
Main Methods:
- Crystallization of the Pap1 DNA-binding domain (DBD) complexed with an ATF/CREB-like non-consensus DNA fragment using the vapor-diffusion technique with polyethylene glycol 6000.
- X-ray diffraction data collection to 2.0 A resolution using a cryo-crystallographic technique.
Main Results:
- Highly ordered crystals of the Pap1 DBD-DNA complex were obtained, belonging to space group R3.
- The crystal structure was determined at 2.0 A resolution, providing detailed insights into the complex's architecture.
Conclusions:
- The determined structure provides a molecular basis for Pap1's recognition of non-consensus DNA sequences.
- This structural information is crucial for understanding Pap1's regulatory mechanisms in gene expression and cellular processes.