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

Nature Methods volume 2, pages 757–762 (2005)Cite this article

Abstract

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome–nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.

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Figure 1: Design of stalled ribosome–nascent chain complexes using the SecM stall sequence.
Figure 2: Visualization of stalled, truncated tailspike and GFP nascent chains.
Figure 3: TβS nascent chains are ribosome-associated.
Figure 4: Stalled ribosome-nascent chain complexes detected by FlAsH binding.
Figure 5: Measurements of mAb 236 binding to stalled tailspike nascent chains and native tailspike trimer.

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Acknowledgements

Plasmid pNH21 was a generous gift of K. Ito. This project was supported by an award from the Clare Boothe Luce Program of the Luce Foundation and the University of Notre Dame. M.S.E. is the recipient of a University of Notre Dame Schmitt Predoctoral Fellowship. M.A.F. was supported by the Deutscher Akademischer Austauschdienst. We thank J. King and T.F. Clarke IV for helpful discussions, P. Huber for use of the gel imaging system, M. Finn for technical assistance, and K. Henderson and R. Nasser for assistance with synthesis of the FlAsH dye.

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  1. Marc-André Frese

    Present address: University of Bielefeld, Faculty of Technology, Cellular Genetics Department, 33501, Bielefeld, Germany

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, 46556, Indiana, USA

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

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Correspondence to Patricia L Clark.

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Evans, M., Ugrinov, K., Frese, MA. et al. Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro. Nat Methods 2, 757–762 (2005). https://doi.org/10.1038/nmeth790

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