Rate of Translation of Natural mRNAs in an Optimizedin VitroSystem

We report results onin vitrotranslation of an mRNA coding for elongation factor TuB which wasin vitrotranscribed from the tufB gene fromEscherichia coli.Translation occurs at a rate of about 10 codons per second, which is close to thein vivorate. 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 measuredKmvalues for EF-Tu and EF-G are 10 and 0.25 μM, respectively. The obtainedkcatandKmvalues were used to estimate the averagekcat/Kmof about 24 × 106s−1M−1for the interaction of individual EF-Tu*GTP*aa–tRNA complexes with ribosomes. The estimatedkcat/Kmvalue for EF-G is 36 × 106s−1M−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 lowerkcat/Kmfor the interaction of EF-Tu*GTP*aa–tRNA complexes with SmP ribosomes.