A mutant T7 RNA polymerase as a DNA polymerase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• T7 RNA polymerase (RNAP) is a key enzyme in transcription.
• RNAP typically discriminates between ribonucleoside triphosphates (rNTPs) for RNA synthesis and deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis.
• The active site of polymerases contains conserved residues that influence substrate selection.

Purpose of the Study:

• To identify and characterize a T7 RNAP mutant with altered substrate specificity.
• To investigate the role of a specific active site tyrosine residue in substrate discrimination.
• To explore the potential applications of such a mutant enzyme.

Main Methods:

• Site-directed mutagenesis of T7 RNAP, specifically targeting Tyr639.
• In vitro enzymatic assays to assess synthesis of RNA, DNA, or mixed polymers using various nucleotide substrates.
• Characterization of mutant enzyme activity, promoter specificity, and template usage.

Main Results:

• A conservative mutation (Tyr639 to Phenylalanine) in T7 RNAP resulted in efficient utilization of dNTPs.
• The Y639F mutant synthesized DNA, RNA, or mixed polymers depending on the available nucleotide mix.
• This mutation did not affect promoter specificity or overall enzyme activity, unlike non-conservative mutations at the same site.
• The mutant retained the ability to use both RNA and DNA templates, exhibiting diverse polymerase activities.

Conclusions:

• Conserved tyrosine residues in polymerases may play a crucial role in sensing substrate and template geometry.
• The Y639F T7 RNAP mutant demonstrates a versatile enzymatic activity profile, adaptable to different nucleic acid synthesis applications.
• This engineered enzyme expands the toolkit for synthetic biology and biotechnology by enabling programmable nucleic acid synthesis.