Rate of translation of natural mRNAs in an optimized in vitro system

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.