Exploration of Arrest-Peptide Sequence Space Reveals Arrest-Enhanced Variants [Protein Synthesis and Degradation]

February 24th, 2015 by Cymer, F., Hedman, R., Ismail, N., von Heijne, G.

Translational arrest peptides (APs) are short stretches of polypeptide that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP, and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that di-proline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation-factor P (EF-P). Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest, and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.