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Molecular and Cellular Biology, July 2008, p. 4227-4239, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00421-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Site-Specific Release of Nascent Chains from Ribosomes at a Sense Codon {triangledown}

Victoria A. Doronina,1 Cheng Wu,2,{dagger} Pablo de Felipe,3 Matthew S. Sachs,2,4,{dagger} Martin D. Ryan,3 and Jeremy D. Brown1*

RNA Biology Group and Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom,1 Department of Environmental & Biomolecular Systems, Oregon Health and Science University, Beaverton, Oregon 97006,2 School of Biology, Centre for Biomolecular Sciences, Biomolecular Sciences Building, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, United Kingdom,3 Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, Oregon 972014

Received 13 March 2008/ Returned for modification 14 April 2008/ Accepted 28 April 2008

"2A" oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific "termination" reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product.

* Corresponding author. Mailing address: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44-191-222-7470. Fax: 44-191-222-7424. E-mail: Jeremy.Brown{at}ncl.ac.uk

{triangledown} Published ahead of print on 5 May 2008.

{dagger} Present address: Department of Biology, Texas A&M University, College Station, TX 77843-3258.

Molecular and Cellular Biology, July 2008, p. 4227-4239, Vol. 28, No. 13
0270-7306/08/$08.00+0     doi:10.1128/MCB.00421-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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