Simulation and analysis of single-ribosome translation.
In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed at the single-molecule level using optical tweezers; this technique allows us to study the kinetics of translation by measuring the lifetime the ribosome spends at each codon. Here, we analyze the data from single-molecule experiments and fit the data with simple kinetic models. We also simulate the translation kinetics based on a multi-step mechanism from ensemble kinetic measurements. The mean lifetimes from the simulation were consistent with our experimental single-molecule measurements. We found that the calculated lifetime distributions were fit in general by equations with up to five rate-determining steps. Two rate-determining steps were only obtained at low concentrations of elongation factors. These analyses can be used to design new single-molecule experiments to better understand the kinetics and mechanism of translation.
Tinoco
Ignacio
I
Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA. Intinoco@lbl.gov
Wen
Jin-Der
JD
D003062:Codon; D003198:Computer Simulation; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D052898:Optical Tweezers; D020653:Peptide Elongation Factor G / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000378:metabolism
D003062:Codon; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D010444:Peptide Elongation Factor Tu