Structural Basis for Translational Stalling by Human Cytomegalovirus and Fungal Arginine Attenuator Peptide

Bhushan, Shashi; Meyer, Helge; Starosta, Agata L.; Becker, Thomas; Mielke, Thorsten; Berninghausen, Otto; Sattler, Michael; Wilson, Daniel N.; Beckmann, Roland

doi:10.1016/j.molcel.2010.09.009

Cited by 115 publications

(121 citation statements)

References 65 publications

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“…3 and 4). These positions are located in the vicinity of the constricted region of the ribosomal tunnel (8), and the effect of tryptophan in these positions may be similar to the tryptophan-binding site seen in the stalled TnaC AP, where bound L-tryptophan interacts with residue Ile Ϫ10 in the AP (9). By simultaneously mutating the three positions Ϫ10 to Ϫ8 to tryptophan, we were able to generate an AP (WWWPPIRGSP) that is able to stall translation even in the presence of the pulling force elicited by 10 aspartic acid residues (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Cymer

Hedman

Ismail

et al. 2015

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Cymer

Hedman

Ismail

et al. 2015

Journal of Biological Chemistry

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“…Bona fide translocating ribosome-Sec61 complexes were obtained using constructs based on the wellcharacterized Lep93 (here referred to as LepT) by Saaf et al 38 These constructs contain glycosylation sites to precisely monitor the translocation state. We modified these constructs to generate and stabilize translocation and insertion intermediates by introducing a C-terminal ribosomal stalling sequence (CMV gp48 upstream open-reading frame) 12 , a streptavidin tag for affinity purification and an haemagglutinin tag for immunodetection. LepT-mRNA was used in a self-made wheatgerm translation system in the presence of PK-RM and signal recognition particle from dog pancreas 15 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

et al. 2014

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In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the proteinconducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

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“…The products were detected with an ANTI-FLAG antibody (F1804, Sigma). The sequence of the human cytomegalovirus (CMV) gp48 uORF2 was used as a positive control for co-translational ribosome stalling 34,35 .…”

Section: Monitoring Of Peptidyl-trna Complexesmentioning

confidence: 99%

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation

Yordanova

Loughran

Zhdanov

et al. 2018

Nature

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Structural Basis for Translational Stalling by Human Cytomegalovirus and Fungal Arginine Attenuator Peptide

Cited by 115 publications

References 65 publications

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

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

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation

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