Kinetics of initiating polypeptide elongation in an IRES-dependent system

• Published in: eLife 2016-06, Vol.5
• Main Authors: Zhang, Haibo, Ng, Martin Y, Chen, Yuanwei, Cooperman, Barry S
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Biochemistry; Biophysics and Structural Biology; Crustacea - virology; Dicistroviridae - classification; Dicistroviridae - genetics; Dicistroviridae - metabolism; Elongation; initial cycles of elongation; Initiation factors; IRES dependent protein synthesis; kinetic mechanism; Kinetics; Paralysis; Peptide Chain Elongation, Translational; Peptide Chain Initiation, Translational; Polypeptides; Polyproteins - genetics; Protein biosynthesis; Protein synthesis; Proteins; rate-limiting translocation; Ribosomes; Ribosomes - metabolism; RNA, Transfer - genetics; RNA, Transfer - metabolism; RNA, Viral - metabolism; tRNA
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.13429

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.