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

Arch Biochem Biophys. 1996 Apr 1;328(1):9-16. doi: 10.1006/abbi.1996.0136.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Base Sequence
  • Cell-Free System
  • Cricetinae
  • Escherichia coli / genetics*
  • Genetic Variation
  • Kinetics
  • Molecular Sequence Data
  • Peptide Chain Elongation, Translational
  • Peptide Elongation Factor G
  • Peptide Elongation Factor Tu / genetics*
  • Peptide Elongation Factors / metabolism
  • Protein Biosynthesis*
  • RNA, Bacterial / metabolism*
  • RNA, Messenger / metabolism*
  • Ribosomes / drug effects
  • Ribosomes / genetics
  • Ribosomes / metabolism
  • Streptomycin / pharmacology


  • Anti-Bacterial Agents
  • Peptide Elongation Factor G
  • Peptide Elongation Factors
  • RNA, Bacterial
  • RNA, Messenger
  • Peptide Elongation Factor Tu
  • Streptomycin