T7 RNA polymerase: conformation, functional groups, and promotor binding

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• T7 RNA polymerase is a crucial enzyme for gene expression in bacteriophage T7.
• Understanding its structural and functional properties is essential for elucidating transcription mechanisms.

Purpose of the Study:

• To investigate the structural stability of T7 RNA polymerase under various conditions.
• To identify key functional residues and their roles in DNA binding and catalysis.
• To analyze the impact of DNA template modification on transcription.

Main Methods:

• Circular dichroism spectroscopy to assess secondary structure.
• Nitrocellulose filter binding assays to measure DNA and nucleotide binding.
• Chemical modification (alkylation, nitration) to probe functional residues.
• Restriction enzyme digestion and gel electrophoresis to analyze transcription products.

Main Results:

• T7 RNA polymerase secondary structure (approx. 12% alpha helix) is stable across a range of conditions.
• A single thiol (-SH) group is essential for catalytic activity but not DNA binding.
• Nitration of surface tyrosyl residues abolishes DNA binding.
• DNA fragmentation by Hpa II alters transcription patterns, producing shorter mRNAs, while promoter regions remain intact.

Conclusions:

• T7 RNA polymerase possesses a stable secondary structure.
• Specific amino acid residues (thiol and tyrosyl) are critical for enzyme function.
• DNA template integrity influences the pattern of RNA synthesis, highlighting the importance of promoter accessibility.