The sarcin–ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit
Abstract
The sarcin–ricin loop (SRL) of 23S rRNA in the large ribosomal subunit is a factor-binding site that is essential for GTP-catalyzed steps in translation, but its precise functional role is thus far unknown. Here, we replaced the 15-nucleotide SRL with a GAAA tetraloop and affinity purified the mutant 50S subunits for functional and structural analysis in vitro. The SRL deletion caused defects in elongation-factor-dependent steps of translation and, unexpectedly, loss of EF-Tu-independent A-site tRNA binding. Detailed chemical probing analysis showed disruption of a network of rRNA tertiary interactions that hold together the 23S rRNA elements of the functional core of the 50S subunit, accompanied by loss of ribosomal protein L16. Our results reveal an influence of the SRL on the higher-order structure of the 50S subunit, with implications for its role in translation.
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Footnotes
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Reprint requests to: Harry F. Noller, Center for Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA 95064, USA; or Department of Molecular, Cell and Developmental Biology, University of California at Santa Cruz, Santa Cruz, CA 95064, USA; e-mail: harry{at}nuvolari.ucsc.edu; fax: (831) 459-3737.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1202108.
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- Received June 4, 2008.
- Accepted June 30, 2008.
- Copyright © 2008 RNA Society
