title: Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.
abstract: Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Wen
authors.initials: W
authors.lastname: Xi
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Shaun
authors.initials: S
authors.lastname: Toomey
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Yueh-Chin
authors.initials: YC
authors.lastname: Chiang
authors.affiliation: Laboratory of Cell Reproduction, Institute of Microbiology of ASCR, Prague, Videnska 1083, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Hasek
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Laue
authors.affiliation: Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, United States of America.
authors.forename: Clyde L
authors.initials: CL
authors.lastname: Denis
mesh_terms: D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D011061:Poly A; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D051137:Selenium-Binding Proteins / Q000378:metabolism
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D046912:Multiprotein Complexes; D012333:RNA, Messenger; D051137:Selenium-Binding Proteins; D011061:Poly A