Article

Kaiser, C.M. et al. Real-time observation of Trigger factor function on translating ribosomes. Nature 444, 455-460

Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Nature (Impact Factor: 42.35). 12/2006; 444(7118):455-60. DOI: 10.1038/nature05225
Source: PubMed

ABSTRACT The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.

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    • "Two algorithms predicting such motifs in the linear (unfolded) sequence were developed earlier: initially, TF-binding sites were predicted as stretches of 8 aa enriched in basic and aromatic residues with a positive net charge (Patzelt et al., 2001) (orange lines in Figure 1 and Figure S1). Later, TF was found to stay associated with nascent chains containing motifs of five or more consecutive amino acids of high mean hydrophobicity (Kaiser et al., 2006) (green lines in Figure 1 and Figure S1). b-lactamase (Figure 1C), ICDH (Figure S1A), MBP, and luciferase contain both types of linear binding motifs. "
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    ABSTRACT: How nascent polypeptides emerging from ribosomes fold into functional structures is poorly understood. Here, we monitor disulfide bond formation, protease resistance, and enzymatic activity in nascent polypeptides to show that in close proximity to the ribosome, conformational space and kinetics of folding are restricted. Folding constraints decrease incrementally with distance from the ribosome surface. Upon ribosome binding, the chaperone Trigger Factor counters folding also of longer nascent chains, to extents varying between different chain segments. Trigger Factor even binds and unfolds pre-existing folded structures, the unfolding activity being limited by the thermodynamic stability of nascent chains. Folding retardation and unfolding activities are not shared by the DnaK chaperone assisting later folding steps. These ribosome- and Trigger Factor-specific activities together constitute an efficient mechanism to prevent or even revert premature folding, effectively limiting misfolded intermediates during protein synthesis.
    Molecular cell 08/2012; 48(1):63-74. DOI:10.1016/j.molcel.2012.07.018 · 14.46 Impact Factor
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    • "An effective functional cooperation apparently exists between TF and the DnaK system in the folding of a group of large multidomain proteins that aggregate substantially only in the absence of both chaperones. These proteins may normally interact sequentially with TF and DnaK during translation or with multiple TF molecules in the absence of DnaK, as shown with large model proteins in vitro (Agashe et al., 2004; Kaiser et al., 2006). Notably, most of these proteins would be unable to interact productively with GroEL, as the capacity of the GroEL/ES folding compartment is limited to proteins up to $60 kDa (Kerner et al., 2005). "
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    ABSTRACT: Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.
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    • "The ribosome binding activity of TF has been extensively characterized in vitro. Although TF binds to non-translating ribosomes with a K D of ~1 μM (Patzelt et al., 2002) and with a mean residence time of 10 to 15 sec (Kaiser et al., 2006), the presence of nascent substrates can increase this affinity up to 30- fold (Rutkowska et al., 2008). In addition, structural analyses of TF in complex with ribosomes suggest that TF forms a protective dome over the tunnel exit (Ferbitz et al., 2004) that could shield nascent chains from degradation by proteases (Hoffmann et al., 2006; Tomic et al., 2006) or improve the efficiency of folding by reducing the speed of folding (Agashe et al., 2004). "
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    ABSTRACT: As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.
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