title: Exploration of the arrest peptide sequence space reveals arrest-enhanced variants.
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.
authors.affiliation: From the Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden and.
authors.forename: Florian
authors.initials: F
authors.lastname: Cymer
authors.affiliation: From the Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden and.
authors.forename: Rickard
authors.initials: R
authors.lastname: Hedman
authors.affiliation: From the Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden and.
authors.forename: Nurzian
authors.initials: N
authors.lastname: Ismail
authors.affiliation: From the Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden and the Science for Life Laboratory Stockholm University, Box 1031, 17121 Solna, Sweden gunnar@dbb.su.se.
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijne
mesh_terms: D000595:Amino Acid Sequence; D001696:Biomechanical Phenomena; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D031121:Mannheimia / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism
chemical_list: D010445:Peptide Elongation Factors; D010455:Peptides; C020746:factor EF-P
keywords: Protein Secretion; Ribosome Function; SecM; Translation; Translation Elongation Factor; Translation Regulation