Abstract
Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA–protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse–chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.
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Acknowledgements
We thank the staff of the TSRI Center for Mass Spectrometry for assistance with mass spectrometry; M. I. Recht, S. C. Agalarov and S. P. Ryder for discussions and technical assistance; the laboratories of D. B. Goodin, S. P. Mayfield and A. Schneemann for use of equipment; and M. J. Fedor, J. D. Puglisi and S. P. Ryder for critically reading the manuscript. This work was supported by a grant from the NIH (to J.R.W.) and by predoctoral fellowships from the NSF and the Skaggs Institute for Chemical Biology (to M.W.T.T.). Author Contributions The experimental work in this manuscript was carried out by M.W.T.T., with advice and support from G.S. and J.R.W.
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Supplementary information
Supplementary Methods
Additional details of the methods used in this study. This file also contains additional references. (DOC 105 kb)
Supplementary Table S1
30S protein binding rates determined by PC/QMS at a range of temperatures and the resulting binding activation energies. (DOC 120 kb)
Supplementary Figure S1
Binding progress curves from PC/QMS and gel mobility shift for the S15:A4 model system. (PDF 2513 kb)
Supplementary Figure S2
30S protein binding progress curves from PC/QMS under standard conditions (as in Fig. 2a). Here the progress curves for the different proteins are shown (PDF 3321 kb)
Supplementary Figure S3
Arrhenius plots of the protein binding rates determined by PC/QMS at a range of temperatures (as in Fig. 4b). Here the Arrhenius plots for the different proteins are shown separately. (PDF 3878 kb)
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Talkington, M., Siuzdak, G. & Williamson, J. An assembly landscape for the 30S ribosomal subunit. Nature 438, 628–632 (2005). https://doi.org/10.1038/nature04261
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DOI: https://doi.org/10.1038/nature04261
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