In Vitro Translation

  • Martin J. Tymms
Part of the Springer Protocols Handbooks book series (SPH)


Transcriptton is a fundamental step in the process of gene expression where the mformation encoded by messenger RNA (mRNA) is translated into a polypeptide sequence. This process requtres rtbosomes, an array of translation factors, a supply of transfer-RNAs loaded with ammo acids, and an energy supply Much of the early discovery and eluctdatton of the fundamental process of translation was carried out using extracts from the prokaryottc bacteria Escherzchza coil, which are capable of supporting the process of protein synthesis. Extracts from special strams of E colz are still used today for the in vitro translation of prokaryotic genes and to a much lesser extent eukaryottc genes Eukaryottc genes need to be modified before they can be translated in prokaryotic translation systems as a consequence of fundamental differences m the ribosome translation systems of eukaryotes and prokaryotes (Fig. 1) In prokaryotes, transcriptton and translation are a coupled process with the rtbosome recogmzing a sitelust 5′ of the mmatron AUG codon (the rtbosome-bmding site, RBS) in the RNA transcript before the process of transcription is complete Eukaryotic mRNAs need to be modified to include a RBS in order to be translated in a prokaryottc translation system In eukaryotes transcrtptton takes place in the nucleus, where the primary transcript, which usually contains mtrons, is processed to yield a mature mRNA that contams a 7-methyl guanosme cap at the 5′ end and in most cases a tail of 100–200 adenosme nucleotides at the 3′ end The mature mRNA moves to the cytoplasm, where tt is recognized by ribosomes by features in the 5′ end of the mRNA that differ from the prokaryottc RBS The eukaryotic rtbosome recognizes the 7-methyl CAP, and sequences around the AUG mttiatton codon are crucial for correct mutation The 7-methyl CAP is crucial for efficient translation in VIVO, but mRNA without a cap can be translated in eukaryotic cell-free translation systems with a reduced efficiency Many RNA viruses have overcome the need for a 7-methyl CAP for efficient translation by mcorporating sequences in the 5′ UTR that allow efficient mitiation of translatton. When these sequences are attached to other nonviral RNAs the need for capping is removed (1).
Fig. 1.

Schematic representation of translation in eukaryotic and prokaryotic organisms. In prokaryotic organisms transcription and translation are a coupled process. In eukaryotes transcription and translation are independent processes in the nucleus and cytoplasm respectively.


Translation System Rabbit Rettculocyte Lysate Eukaryotic mRNA Protein Truncation Test Vitro Translation 
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Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

  • Martin J. Tymms
    • 1
  1. 1.Instttute for Reproductton and DevelopmentMonash Medical CenterAustralia

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