Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Abstract

Background:The stalling efficiency of translational arrest peptides (APs) is sensitive to mechanical pulling forces on the nascent chain. Results: We identify new APs with enhanced stalling efficiency. Conclusion: Mechanical pulling forces reduce stalling induced by diproline stretches in efp Ϫ cells. Significance:Our results provide new insights into AP-induced translational stalling and offer new in vivo force sensors.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.During protein synthesis, the nascent polypeptide chain moves through the ϳ100-Å-long exit tunnel in the large ribosomal subunit (1). Strong interactions between the nascent chain and the tunnel might adversely affect protein synthesis and can even lead to complete blockage of the ribosome, a mechanism exploited by many antibiotics (2). It has therefore been postulated that the ribosome exit tunnel has a "Teflonlike" surface that minimizes interactions with the nascent chain to avoid adverse effects on translation (1, 3).Nevertheless, some nascent chain segments are able to interact with the ribosomal exit tunnel in ways that block or slow down translation (4, 5). In bacteria, such translational arrest peptides (APs) 2 are often used to regulate the translation of downstream open reading frames in polycistronic mRNAs (6, 7). APs interact with distinct ribosomal RNA and protein components within the ribosomal exit tunnel (8), inducing conformations at the ribosome active site that can block the peptidyl transfer reaction (9 -11).In the case of the AP-containing SecM protein in Escherichia coli, the arrest of nascent chain elongation can be overcome by the activity of the motor protein SecA, which presumably breaks the AP-tunnel interactions by mechanically pulling on the nascent chain (12). On the basis of this notion, we hypothesized that...

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems

“…The extension consists of a region rich in Gly and Ser followed by the SecM (FSTPVWISQAQGIRAGP)21 or the SecMstr AP (FSTPVWI WWWPR IR GP P), where the underlined residues were changed according to reference24. The SecM AP induces translational arrest, by shifting the linkage between the nascent chain and the peptidyl-tRNA by 2 Å1819.…”

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

“…We also recorded the f FL and f TR profiles for wild-type S6 and the supercharged S6 −16 variant using the stronger SecM(Ms) AP (38). The f TR profile for wild-type S6 mirrors the f FL profile, starting to increase at L ≈ 27 residues and plateauing at L max ≈ 32 residues (Fig.…”

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

“…very stable variant S6-16 ; in this case, f FL ≥ 0.7 for all L values despite the low stability of the protein, and the profile has a broad maximum at L max ≈ 48 residues,Fig. 4f.We also recorded the f TR profiles for wildtype S6 and the supercharged S6 -16 variant, using the stronger SecM(Ms) arrest peptide24 . The f TR profile for wildtype S6 mirrors the f FL profile, starting to increase at L ≈ 27 residues and plateauing at L ≈ 32 residues,Fig.…”