Non-programmed transcriptional frameshifting is common and highly RNA polymerase type-dependent

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Gene expression relies on accurate transcription and high-quality protein products.
• Nucleotide insertion/deletion (indel) during transcription, known as slippage, can occur at homopolymer sequences (A/T) and reduce expression efficiency.
• Comparative studies on slippage propensity between Escherichia coli and T7 bacteriophage RNA polymerases (RNAPs) were lacking.

Purpose of the Study:

• To systematically investigate and compare the transcriptional slippage capabilities of T7 phage and E. coli RNAPs.
• To evaluate the influence of homopolymer length, template type, and frameshift directionality on RNAP slippage.
• To assess the impact of slippage on gene expression and protein product fidelity.

Main Methods:

• Utilized out-of-frame gfp reporter genes fused to various A/T homopolymeric sequences.
• Examined RNAP-specific expression profiles under potent promoters (P_araBAD for E. coli, φ10 for T7).
• Assessed indel-dependent phenotypic changes to quantify slippage events.

Main Results:

• T7 RNAP demonstrates significant frameshifting potential, slipping on as few as three adenine or four thymidine residues.
• E. coli RNAP shows lower slippage ability, requiring at least seven thymidine or more adenine residues.
• T7 RNAP insertion slippage favors poly(A) over poly(T), while E. coli RNAP exhibits a bias towards poly(T).

Conclusions:

• Intrinsic RNAP slippage involves trade-offs between accuracy, speed, and processivity.
• T7 RNAP's higher inclination for slippage may aid bacteriophage adaptation.
• Bacterial expression systems with proofreading mechanisms are recommended for biotechnological applications requiring high fidelity.