Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Evans, Michael S.; Ugrinov, Krastyu G.; Frese, Marc-André; Clark, Patricia L.

doi:10.1038/nmeth790

Cited by 69 publications

(84 citation statements)

References 31 publications

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“…To produce arrested SH3 and m10 polypeptides exposing the entire SH3 or m10 domains outside the ribosomal exit tunnel, we introduced a C-terminal 41-aa linker. This linker carried the SecM stalling sequence known to arrest translation and was long enough to extend through the entire length of the ribosomal peptide tunnel (27,28).…”

Section: Resultsmentioning

confidence: 99%

Trigger Factor forms a protective shield for nascent polypeptides at the ribosome

Merz¹,

Rutkowska²,

Deuerling³

et al. 2007

GBM Annual Spring Meeting Mosbach 2007

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In prokaryotes, the ribosome-associated Trigger Factor is the first chaperone newly synthesized polypeptides encounter when they emerge from the ribosomal exit tunnel. The effects that Trigger Factor exerts on nascent polypeptides, however, remain unclear. Here we analyzed the potential of the Trigger Factor to shield nascent polypeptides at the ribosome. A set of arrested nascent polypeptides differing in origin, size, and folding status were synthesized in an Escherichia coli-based in vitro transcription/translation system and tested for sensitivity to degradation by the unspecific protease proteinase K. In the absence of Trigger Factor, nascent polypeptides exposed outside the ribosomal exit tunnel were rapidly degraded unless they were folded into a compact domain. The presence of Trigger Factor, as well as a Trigger Factor fragment lacking its peptidyl-prolyl isomerase domain, counteracted degradation of all unfolded nascent polypeptides tested. This protective function was specific for ribosome-tethered Trigger Factor, since neither non-ribosomal Trigger Factor nor the DnaK system, which cooperates with Trigger Factor in the folding process in vivo, revealed a comparable efficiency in protection. Furthermore, shielding by Trigger Factor was not restricted to short stretches of nascent chains but was evident for large, non-native nascent polypeptides exposing up to 41 kDa outside the ribosome. We suggest that Trigger Factor supports productive de novo folding by shielding nascent polypeptides on the ribosome thereby preventing untimely degradation or aggregation processes. This protected environment provided by Trigger Factor might be particularly important for large multidomain proteins to fold productively into their native states.Cells employ a large arsenal of molecular chaperones to promote the folding of newly synthesized proteins in the cytosol (1-3). At the forefront are chaperones, which transiently bind to ribosomes and thereby are optimally positioned to associate with emerging nascent polypeptides. In eubacteria, Trigger Factor (TF) 3 binds to ribosomes near the exit of the peptide tunnel through major contacts to L23 (4 -7) and interacts co-translationally with nascent polypeptides (8, 9). Downstream of TF, the DnaK-DnaJ-GrpE (referred to as KJE) and GroELGroES chaperone systems assist further folding steps to the native state (2, 10). Together these chaperones form a folding network of considerable robustness. Thus, the lack of TF can be compensated by activity of the KJE system, while simultaneous deletion of dnaK and the TF encoding tig gene results in synthetic lethality at Ն30°C and massive protein aggregation (11-13).TF is a three-domain protein of 48 kDa that adopts an unusual extended conformation (Fig. 1A) (5, 14). The N-terminal domain builds the "tail" of the molecule and harbors the "TF-signature motif," which mediates ribosome docking (4). A peptidyl-prolyl cis/trans isomerase (PPIase) domain with structural homology to FK506-binding proteins (9, 16) is located at the other end of ...

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Section: Resultsmentioning

confidence: 99%

Trigger Factor forms a protective shield for nascent polypeptides at the ribosome

Merz¹,

Rutkowska²,

Deuerling³

et al. 2007

GBM Annual Spring Meeting Mosbach 2007

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“…Once translated, this 18-residue peptide interacts with the exit tunnel of the 50S subunit leading to translation stalling (Nakatogawa and Ito 2001;Yap and Bernstein 2009). The SecM arrest sequence has been shown to be an effective method of anchoring a mature protein to the ribosome immediately following translation in both single-molecule and bulk experiments (Evans et al 2005;Uemura et al 2008). The stalled 70S-SNAP f complex has a half-life of ∼60 min (Evans et al 2005;Tsai et al 2014), so turnover is still observed in longer time courses.…”

Section: Designing the Snap Assaymentioning

confidence: 99%

“…The SecM arrest sequence has been shown to be an effective method of anchoring a mature protein to the ribosome immediately following translation in both single-molecule and bulk experiments (Evans et al 2005;Uemura et al 2008). The stalled 70S-SNAP f complex has a half-life of ∼60 min (Evans et al 2005;Tsai et al 2014), so turnover is still observed in longer time courses. The arrest sequence has two functions in the SNAP assay.…”

Section: Designing the Snap Assaymentioning

confidence: 99%

A simple real-time assay for in vitro translation

Capece

Kornberg

Petrov

et al. 2014

RNA

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A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O 6 -alkylguanine DNA O 6 -alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC 50 ) of three common macrolide antibiotics.

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“…that may affect the binding of TF to ribosome, it is essential to investigate RNCs harboring defined states of nascent chains, and hence to arrest the translation process at selected stages. For this purpose, we improved a method that allows the stalling of nascent chains in E. coli cells followed by the isolation of quantitative amounts of stalled RNCs (19,20). To induce translational arrest, we genetically fused the 3Ј-end of the open reading frames of target genes to a linker encoding both the SecM peptide, which induces translational arrest (21), and additional amino acids (aa) to allow the complete exposure of the target protein at the ribosomal exit site (Fig.…”

Section: Design Of Arrested Nascent Chain Constructs-see Supplementalmentioning

confidence: 99%

Dynamics of Trigger Factor Interaction with Translating Ribosomes

Rutkowska

Mayer

Hoffmann

et al. 2008

Journal of Biological Chemistry

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In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.

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Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Cited by 69 publications

References 31 publications

Trigger Factor forms a protective shield for nascent polypeptides at the ribosome

Trigger Factor forms a protective shield for nascent polypeptides at the ribosome

A simple real-time assay for in vitro translation

Dynamics of Trigger Factor Interaction with Translating Ribosomes

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