Single crystals of long DNA molecules

Area of Science:

• Molecular Biology
• Structural Biology
• Biophysics

Background:

• Transcription factor IIIA (TFIIIA) is crucial for ribosomal RNA gene expression in Xenopus.
• Crystallization of long DNA fragments, especially those containing protein binding sites, is challenging.
• Understanding DNA structure at binding sites is key to deciphering gene regulation mechanisms.

Purpose of the Study:

• To crystallize and determine the high-resolution structure of long DNA molecules encompassing the TFIIIA binding site.
• To investigate the structural characteristics of DNA within these crystals and compare them to DNA in solution.
• To explore how crystal packing influences DNA conformation.

Main Methods:

• Growth of single crystals of DNA molecules (54 and 65 base pairs).
• X-ray diffraction analysis to determine crystal structure and order.
• Circular Dichroism (CD) spectroscopy to analyze DNA structure in solution.

Main Results:

• Successfully crystallized significantly longer DNA molecules (54 and 65 bp) than previously achieved.
• X-ray diffraction data revealed ordered crystals diffracting to 9 Å resolution.
• DNA molecules formed layered structures with interdigitated phosphate backbones in major and minor grooves; average structure appeared B-like, contrasting with solution CD spectra indicating an A/B-intermediate form.

Conclusions:

• Long DNA crystals containing the TFIIIA binding site can be successfully grown and analyzed.
• The observed B-like DNA structure in crystals may be an artifact of tight intermolecular packing.
• Discrepancies between crystal and solution structures highlight the importance of considering environmental influences on DNA conformation.