# Translation‐Promoting Effects of RNA Template Overhangs in the Absence of Ribosomes

## TL;DR

This paper shows how RNA overhangs can control and enhance ribosome-free translation of peptides.

## Contribution

The study introduces triplex-forming RNA overhangs that regulate and boost ribosome-free translation efficiency.

## Key findings

- Triplex-forming RNA overhangs increase ribosome-free translation yields up to 40-fold.
- Mixed anhydrides between amino acid residues and phosphates improve peptide coupling efficiency.
- pH-dependent triplex folding enables inducible translation without biomacromolecules.

## Abstract

Translation requires a complex set of biomacromolecules. How it evolved is an unsolved problem. Ribosome‐free, single‐nucleotide translation via coupling of 3′‐aminoacylated RNA to 5′‐phosphoramidate‐linked RNAs was shown for very short peptides. It was unclear how this process could be induced at a specific locus, regulated, and expanded. Here we show that a triplex‐forming template overhang accelerates ribosome‐free translation and increases its yield. An NMR‐monitored model study suggested that mixed anhydrides between carboxylates of amino acid residues and phosphates can contribute to efficient peptide coupling, strengthening proximity effects. Folding into triplexes at pH 6 enhances the template effect, and unfolding at pH 8 suppresses it, suggesting a simple way of achieving “inducible translation” in a system devoid of biomacromolecules. Under mild acidic conditions, doubly RNA‐linked peptides up to octamers were formed in near‐quantitative yield. Taken together, our data suggests that the growth of template strands not only allows for encoding more genetic information, but may also enhance translational fitness.

Ribosome‐free translation of an RNA sequence into a peptide sequence can be controlled by the folding of the template strand. Overhang sequences forming a triplex structure with the templating region and the peptidoyl RNA in pH‐dependent manner increase yields up to 40‐fold through entropic effects and mixed anhydride formation, allowing for controlled, locus‐specific translation.