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Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Nature volume 455, pages 1268–1272 (2008)Cite this article

Abstract

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth1,2. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting3 was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5′, the central and the 3′ domains require 2–200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA–protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.

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Figure 1: Time-resolved X-ray footprinting of 30S ribosome assembly.
Figure 2: Simultaneous folding of 16S domains.
Figure 3: Stepwise assembly of RNA and protein interactions.
Figure 4: Ribosomal proteins interact with the rRNA in stages.

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Acknowledgements

We thank R. Moss, A. Cukras, L. Cochella and R. Green for help with ribosome preparation and peptidyl transferase assays, P. Fleming for help with Calc-Surf software, and S. Gupta, M. Sullivan and M. Brenowitz for help with X-ray footprinting. This work was supported by the National Institutes of Health (NIH; GM60819). The NSLS X28C and the Center for Synchrotron Biosciences are supported by NIH P41-EB0001979.

Author Contributions T.A. performed the experiments, analysed the data and prepared the figures; D.L.B. analysed protections in the 3′ minor domain; and S.A.W. prepared the figures and wrote the paper.

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Author notes

  1. Tadepalli Adilakshmi & Deepti L. Bellur

    Present address: Present address: Weis Center for Research, Geisinger Medical Center, 100 North Academy Avenue, Danville, Pennsylvania 17822, USA (T.A.); Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637, USA (D.L.B).,

  2. Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218-2685, USA

Authors and Affiliations

  1. T.C. Jenkins Department of Biophysics, and,

    Sarah A. Woodson

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  1. Tadepalli Adilakshmi
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  3. Sarah A. Woodson
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Correspondence to Sarah A. Woodson.

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Adilakshmi, T., Bellur, D. & Woodson, S. Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly. Nature 455, 1268–1272 (2008). https://doi.org/10.1038/nature07298

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