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      Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP

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          Abstract

          Ribosomes must dissociate into subunits in order to begin protein biosynthesis. The enzymes that catalyze this fundamental process in eukaryotes remained unknown. Here, we demonstrate that eukaryotic translocase, eEF2, which catalyzes peptide elongation in the presence of GTP, dissociates yeast 80S ribosomes into subunits in the presence of ATP but not GTP or other nucleoside triphosphates. Dissociation was detected by light scattering or ultracentrifugation after the split subunits were stabilized. ATP was hydrolyzed during the eEF2-dependent dissociation, while a non-hydrolyzable analog of ATP was inactive in ribosome splitting by eEF2. GTP inhibited not only ATP hydrolysis but also dissociation. Sordarin, a fungal eEF2 inhibitor, averted the splitting but stimulated ATP hydrolysis. Another elongation inhibitor, cycloheximide, also prevented eEF2/ATP-dependent splitting, while the inhibitory effect of fusidic acid on the splitting was nominal. Upon dissociation of the 80S ribosome, eEF2 was found on the subunits. We propose that the dissociation activity of eEF2/ATP plays a role in mobilizing 80S ribosomes for protein synthesis during the shift up of physiological conditions.

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          The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection.

          To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5'-end of an unstructured 5'-untranslated region (5'-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5'-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5'-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5'-proximal AUG triplets located only 1, 2, and 4 nt from the 5'-end of mRNA.
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            Polyamines: mysterious modulators of cellular functions.

            In recent years the functions of polyamines (putrescine, spermidine, and spermine) have been studied at the molecular level. Polyamines can modulate the functions of RNA, DNA, nucleotide triphosphates, proteins, and other acidic substances. A major part of the cellular functions of polyamines can be explained through a structural change of RNA which occurs at physiological concentrations of Mg(2+) and K(+) because most polyamines exist in a polyamine-RNA complex within cells. Polyamines were found to modulate protein synthesis at several different levels including stimulation of special kinds of protein synthesis, stimulation of the assembly of 30 S ribosomal subunits and stimulation of Ile-tRNA formation. Effects of polyamines on ion channels have also been reported and are gradually being clarified at the molecular level. Copyright 2000 Academic Press.
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              The molecular mechanics of eukaryotic translation.

              Great advances have been made in the past three decades in understanding the molecular mechanics underlying protein synthesis in bacteria, but our understanding of the corresponding events in eukaryotic organisms is only beginning to catch up. In this review we describe the current state of our knowledge and ignorance of the molecular mechanics underlying eukaryotic translation. We discuss the mechanisms conserved across the three kingdoms of life as well as the important divergences that have taken place in the pathway.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                July 2007
                22 June 2007
                22 June 2007
                : 35
                : 14
                : 4597-4607
                Affiliations
                1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107 and 2Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA
                Author notes
                *To whom correspondence should be addressed.+1 215 503 6547+1 215 923 7343 Hideko.Kaji@ 123456jefferson.edu
                Article
                10.1093/nar/gkm468
                1950535
                17586816
                e02bacbf-3cab-4797-a1f0-48588ca4e8fb
                © 2007 The Author(s)

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Nucleic Acid Enzymes

                Genetics
                Genetics

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