RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA-dependent T7 RNA polymerase (T7 RNAP) is a widely used enzyme for in vitro transcription and gene expression.
• Understanding the fidelity of T7 RNAP is crucial for accurate mRNA synthesis and downstream applications.

Purpose of the Study:

• To analyze the transcriptional fidelity of T7 RNAP using Next Generation Sequencing (NGS).
• To investigate the impact of T7 RNAP-generated mRNA polymorphism on gene expression and protein function.

Main Methods:

• Utilized Next Generation Sequencing (NGS) to analyze mRNA pools generated by T7 RNAP.
• Quantified nucleotide misincorporations and insertions in homopolymer regions.
• Assessed the functional consequences of mRNA sequence variations on protein production.

Main Results:

• T7 RNAP demonstrated a notable level of template-dependent transcriptional infidelity.
• Nucleotide misincorporations (0.20%) and insertions (0.089%) in mRNA, particularly in A/T-rich tracts, led to significant epigenetic effects on gene expression.
• Observed sequence-dependent rescue of frameshifting mutants and recovery of wild-type phenotypes.
• Generated heterogeneous protein pools with varied functionality but similar molecular mass.

Conclusions:

• Transcriptional infidelity in T7 RNAP, despite its low frequency, has a disproportionately large impact on gene expression.
• This infidelity can lead to the production of diverse protein variants, potentially enhancing evolutionary adaptability.
• The study highlights the functional significance of errors in highly efficient RNA polymerases.