iTP-seq: a scalable profiling workflow to characterize bacterial translation landscapes in vitro

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Translation rates influence protein expression and folding.
• Ribosome profiling (Ribo-seq) is a common in vivo method for studying protein synthesis.
• Existing methods may require prior knowledge of sequenced genomes.

Purpose of the Study:

• To present a robust protocol for inverse toeprinting coupled to next-generation sequencing (iTP-seq).
• To demonstrate iTP-seq's utility in studying context-dependent translation inhibitors, like antibiotics.
• To establish iTP-seq as a complementary in vitro method to Ribo-seq.

Main Methods:

• iTP-seq involves generating ribosome-protected mRNA fragments (inverse toeprints) using RNase R.
• Deep sequencing of these fragments determines leading ribosome positions and translated upstream regions.
• The protocol is standardized for inverse toeprint generation, amplification, and sequencing.

Main Results:

• iTP-seq provides codon-resolution mapping of ribosome positions on mRNA.
• The method requires no a priori knowledge of translated sequences, allowing customizable transcript libraries.
• The workflow can be completed in approximately 10 days.

Conclusions:

• iTP-seq is a scalable and versatile in vitro method for characterizing bacterial translation landscapes.
• This technique can be integrated into broader workflows to study translation inhibitors.
• iTP-seq complements in vivo methods like Ribo-seq, offering a customizable approach to protein synthesis research.