Abstract
Translational arrest peptides (APs) are short stretches of polypeptides 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 diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor 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.
Highlights
The stalling efficiency of translational arrest peptides (APs) is sensitive to mechanical pulling forces on the nascent chain
Just how much pulling force can they be made to withstand? Which residues in an AP are the most critical to the arrest potency and, interact most strongly with the ribosomal tunnel? Here we show that the interactions between the SecM(Ms-Sup1) AP and the ribosomal exit tunnel can be tuned to considerably increase the strength of stalling
We demonstrate that diproline stretches, which induce stable translational stalling in elongation factor P (EF-P) deletion strains, effectively function as very short force-sensitive arrest peptides
Summary
The stalling efficiency of translational arrest peptides (APs) is sensitive to mechanical pulling forces on the nascent chain. It is 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 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. In E. coli cells lacking elongation factor P (EF-P), diproline stretches in nascent polypeptide chains can cause ribosome stalling [3] To test whether this kind of stalling mechanism is sensitive to pulling forces, we measured the strength of the translational arrest induced by diproline stretches with different flanking residues in an efpϪ strain, demonstrating their possible use as exceptionally short in vivo force sensors
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