Phasing RNA polymerase II using intrinsically bound Zn atoms: an updated structural model.

Area of Science:

• Structural Biology
• Biochemistry
• X-ray Crystallography

Background:

• Macromolecular assemblies are crucial for cellular functions.
• Phasing large protein complexes for structural determination is challenging.
• Zinc atoms are known to bind to various proteins.

Purpose of the Study:

• To investigate the use of intrinsically bound zinc atoms for phasing large macromolecular assemblies.
• To determine the phasing effectiveness of zinc atoms for RNA polymerase II (Pol II).
• To obtain an unbiased electron density map of Pol II for structural refinement.

Main Methods:

• Multi-wavelength anomalous diffraction (MAD) experiments were performed.
• Phasing was achieved using intrinsically bound zinc atoms within the Pol II structure.
• Computational experiments with simulated MAD data were conducted.

Main Results:

• A phasing effectiveness of 570 amino acids per zinc atom was achieved for Pol II.
• The experimental electron density map confirmed the existing crystallographic model and revealed new structural regions.
• The fork loop-1 element was observed to be ordered in the absence of nucleic acids.
• Simulated data showed that one zinc site can phase up to 1100 amino acids.

Conclusions:

• Intrinsically bound zinc atoms are effective for phasing large macromolecular assemblies like Pol II.
• This method provides high-quality electron density maps, enabling model refinement and discovery of novel structural features.
• The ordered fork loop-1 element offers insights into transcription ternary complex stability and release mechanisms.