A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits

T. Martin Schmeing, Amy C. Seila, Jeffrey L. Hansen, Betty Freeborn, Juliane K. Soukup, Stephen A. Scaringe, Scott A. Strobel, Peter B. Moore, Thomas A. Steitz

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Abstract

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3′ OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

Original languageEnglish (US)
Pages (from-to)225-230
Number of pages6
JournalNature Structural Biology
Volume9
Issue number3
DOIs
StatePublished - Jan 1 2002

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All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry
  • Genetics

Cite this

Schmeing, T. M., Seila, A. C., Hansen, J. L., Freeborn, B., Soukup, J. K., Scaringe, S. A., Strobel, S. A., Moore, P. B., & Steitz, T. A. (2002). A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits. Nature Structural Biology, 9(3), 225-230. https://doi.org/10.1038/nsb758