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

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.

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Acknowledgements

We thank F.M. Richards, A.K. Oylere, D. Kitchen and S.P. Ryder for advice concerning experimental design and execution; D.J. Klein, A. Ray and A.A. Szewczak for helpful discussions; S. Kamtekar for critical reading of the manuscript; and J. Wang and A.R. Curran for assistance with computation. We are indebted to A. Joachimiak and the staff of 19-ID at the Advanced Photon Source (Argonne National Laboratory). This research was supported by NIH grants to T.A.S., P.B.M. and S.A.S., and an Agouron Institute grant to T.A.S. and P.B.M.

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The authors declare no competing financial interests.

Correspondence to Thomas A. Steitz.

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Figure 1: Schematic of the modified fragment assay.
Figure 2: Demonstration of peptide bond formation catalyzed by 50S subunits in the absence of organic co-solvent.
Figure 3: Stereo view of the difference electron density map showing the formation of the product bound in the A-site.
Figure 4: Structure of the new fragment reaction products bound to the ribosome.
Figure 5: Activity of H. marismortui 50S ribosomal subunits in crystallized form.
Figure 6: A new step in the hybrid states model of protein synthesis.