title: Kinetics of initiating polypeptide elongation in an IRES-dependent system.
abstract: The intergenic IRES of Cricket Paralysis Virus (CrPV-IRES) forms a tight complex with 80S ribosomes capable of initiating the cell-free synthesis of complete proteins in the absence of initiation factors. Such synthesis raises the question of what effect the necessary IRES dissociation from the tRNA binding sites, and ultimately from all of the ribosome, has on the rates of initial peptide elongation steps as nascent peptide is formed. Here we report the first results measuring rates of reaction for the initial cycles of IRES-dependent elongation. Our results demonstrate that 1) the first two cycles of elongation proceed much more slowly than subsequent cycles, 2) these reduced rates arise from slow pseudo-translocation and translocation steps, and 3) the retarding effect of ribosome-bound IRES on protein synthesis is largely overcome following translocation of tripeptidyl-tRNA. Our results also provide a straightforward approach to detailed mechanistic characterization of many aspects of eukaryotic polypeptide elongation.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, United States.
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, United States.
authors.forename: Martin Y
authors.initials: MY
authors.lastname: Ng
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, United States.
authors.forename: Yuanwei
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, United States.
authors.forename: Barry S
authors.identifier: 0000-0001-6989-9788
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D000818:Animals; D003445:Crustacea / Q000821:virology; D056629:Dicistroviridae / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D020815:Polyproteins / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
chemical_list: D020815:Polyproteins; D012367:RNA, Viral; D012343:RNA, Transfer
keywords: IRES dependent protein synthesis; biochemistry; biophysics; initial cycles of elongation; kinetic mechanism; none; rate-limiting translocation; structural biology