title: Rate of translation of natural mRNAs in an optimized in vitro system.
abstract: We report results on in vitro translation of an mRNA coding for elongation factor TuB which was in vitro transcribed from the tufB gene from Escherichia coli. Translation occurs at a rate of about 10 codons per second, which is close to the in vivo rate. Protein elongation obeys Michaelis-Menten kinetics with respect to the concentrations of the elongation factors EF-Tu and EF-G in the translation system. The measured K(m) values for EF-Tu and EF-G are 10 and 0.25 microM, respectively. The obtained k(cat) and K(m) values were used to estimate the average k(cat)/K(m) of about 24 x 10(6) s-1 M-1 for the interaction of individual EF-Tu*GTP*aa-tRNA complexes with ribosomes. The estimated k(cat)/K(m) value for EF-G is 36 x 10(6) s-1 M-1. We have also studied translation with a "hyperaccurate" ribosome variant that is pseudodependent on streptomycin (SmP). We have found that SmP ribosomes translate the TuB mRNA significantly slower than wild-type ribosomes do. This is mainly due to a threefold lower k(cat)/K(m) for the interaction of EF-Tu*GTP*aa-tRNA complexes with SmP ribosomes.
authors.affiliation: Department of Molecular Biology, BMC, Uppsala, Sweden.
authors.forename: M Y
authors.initials: MY
authors.lastname: Pavlov
authors.forename: M
authors.initials: M
authors.lastname: Ehrenberg
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002474:Cell-Free System; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics*; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D010445:Peptide Elongation Factors / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013307:Streptomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D010444:Peptide Elongation Factor Tu; D013307:Streptomycin