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Real-time observation of trigger factor function on translating ribosomes

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 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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Figure 1: TF undergoes structural rearrangements upon binding to ribosomes.
Figure 2: TF remains associated with the nascent chain upon leaving the ribosome.
Figure 4: Chain-length dependence of TF interaction with titin I27 and model of TF function.
Figure 3: The nature of the nascent polypeptide influences its interaction with TF.

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Acknowledgements

We thank A. Johnson for advice regarding assessment of fluorophore mobility, A. Bracher for help in designing the ΔPPIase construct and advice during building of the TF dimer model, M. Kerner for his help with bioinformatics analyses, R. Boteva and K. Chakroborty for discussion, P. Genevaux and C. Georgopoulos for the GP 367 E. coli strain and P. Schultz for reagents for in vivo crosslinking. J.M.B. was supported by a fellowship from the International Human Frontier Science Program Organization. Support by the Ernst Jung Foundation and the European Union is also acknowledged. Author Contributions F.U.H. was the project leader. C.M.K. and J.M.B. were responsible for experimental and project design. C.M.K. performed most of the experiments. V.R.A. made conceptual contributions and H.-C.C., V.R.A., S.K.L., S.A.E., M.H.-H. and J.M.B. performed some of the experiments. F.U.H. and J.M.B. wrote the manuscript.

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Correspondence to José M. Barral.

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Kaiser, C., Chang, HC., Agashe, V. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006). https://doi.org/10.1038/nature05225

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