Recombinant Thermus aquaticus RNA polymerase for structural studies

Area of Science:

• Structural biology
• Bacterial transcription
• Enzymology

Background:

• Structural studies of bacterial RNA polymerases (RNAPs) from Thermus aquaticus (Taq) and Thermus thermophilus (Tth) are crucial for understanding transcription.
• Previous limitations included reliance on difficult-to-purify endogenous enzymes and lack of genetic manipulation options.
• Existing recombinant systems did not yield RNAP suitable for crystallographic studies.

Purpose of the Study:

• To develop functional recombinant systems for preparing Taq RNAP suitable for crystallographic studies.
• To investigate the role of the Taq beta' non-conserved domain (NCD) in RNAP structure and function.
• To enable structural analysis of genetically modified RNAPs.

Main Methods:

• Generation of recombinant Thermus aquaticus RNA polymerase (Taq RNAP) with deletions in the beta' non-conserved domain (NCD).
• Co-expression and assembly of modified Taq RNAP in a heterologous host (Escherichia coli).
• Crystallographic studies of the recombinant Taq RNAP mutants.
• Biochemical characterization of beta'NCD deletion mutants.

Main Results:

• Successfully produced recombinant Taq RNAP, including variants with beta'NCD deletions, suitable for crystallographic analysis.
• Demonstrated that the beta'NCD is essential for efficient binding of the sigma subunit.
• Preliminary structural analysis revealed a novel conformation of the beta-flap in a recombinant mutant.

Conclusions:

• The developed recombinant systems overcome previous limitations, enabling structural studies of genetically manipulated Taq RNAP.
• The beta'NCD plays a significant role in sigma subunit interaction.
• Structural insights into the beta-flap conformation expand our understanding of RNAP flexibility.