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Nucleic Acids Res. 2014 Mar;42(5):3419-35. doi: 10.1093/nar/gkt1295. Epub 2013 Dec 13.

Functional domains of the 50S subunit mature late in the assembly process.

Author information

1
Department of Biochemistry and Biomedical Sciences and MG. DeGroote Institute for Infectious Diseases Research, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S4K1, Canada, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA and Department of Integrative Structural and Computational Biology, Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Abstract

Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.

PMID:
24335279
PMCID:
PMC3950693
DOI:
10.1093/nar/gkt1295
[Indexed for MEDLINE]
Free PMC Article

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