Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors

Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, Department of Chemistry, Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL 33458, Department of Pharmacology and Molecular Sciences, School of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan and Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Nucleic Acids Research (Impact Factor: 8.81). 10/2012; 41(1). DOI: 10.1093/nar/gks958
Source: PubMed

ABSTRACT Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3's known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

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