Advertisement

In Vitro Translation

  • Martin J. Tymms
Protocol
  • 1k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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.

Keywords

Translation System Rabbit Rettculocyte Lysate Eukaryotic mRNA Protein Truncation Test Vitro Translation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Galhe, D R, Sleat, D E, Watts, J W, Turner, P C, and Wilson, T in (1987) The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vlvo Nucleic Acids Res 15,3257–3273CrossRefGoogle Scholar
  2. 2.
    Lesley, S A (1995) Preparation and use of E co11 S-30 extracts, in Methods zn Molecular Bzology, vol 37 In Vztro Transmptlon and Translation Protocols (Tymms, M J, ed ), Humana, Totowa, NJ, pp 265–278CrossRefGoogle Scholar
  3. 3.
    Beckler, G S, Thompson, D, and Van Oosbree, T (1995) In vitro translation using rabbit retlculocyte lysate, in Methods w Molecular Bzology, vol 37 In Vitro Transcnptlon and Translation Protocols (Tymms, M J, ed ), Humana, Totowa, NJ, pp 215–232CrossRefGoogle Scholar
  4. 4.
    Van Herwynen, J F and Beckler, G S ( 1995) Translation using a wheat-germ extract, in Methods in Molecular Biology, vol 37 In Vitro Transcription and Translation Protocols (Tymms, M J, ed ), Humana, Totowa, NJ, pp 245–251CrossRefGoogle Scholar
  5. 5.
    Matthews, G in and Colman, A (1995) The Xenopus egg extract translation system, in Methods in Molecular Biology, vol 37 In Vitro Transcrlptlon and Translation Protocols (Tymms, M J, ed ), Humana, Totowa, NJ, pp 199–214CrossRefGoogle Scholar
  6. 6.
    Cenottl, A and Colman, A (1995) mRNA translation in Xenopus oocytes, in Methods in Molecular Bzology, vol 37 In Vitro Transcription and Translation Protocols (Tymms, M J, ed), Humana, Totowa, NJ, pp 151–178Google Scholar
  7. 7.
    Craig, D, Howell, M T, Gibbs, C L, Hunt, T, and Jackson, R J (1992) Plasmld cDNAdirected protein synthesis in a coupled eukaryotlc in vitro transcription-translation systemNuclezc Acids Res 20,4987–4995CrossRefGoogle Scholar
  8. 8.
    Matlas, W G, Bomm, M, and Creppy, E E (1996) Inhlbltlon of protein synthesis in a cell-free system and Vero cells by okadalc acid, a dlarrhetlc shellfish toxin J Toxzcol Envwon Health 48,309–317CrossRefGoogle Scholar
  9. 9.
    Oka, T, TSUJI, H, Noda, C, Sakal, K, Hong, Y in, Suzuki, I, Munoz, S, and Naton, Y (1995) Isolation and characterlzatlon of a novel perchlorlc acid-soluble protein mhlblting cell-free protem synthesis J Blol Chem 270,30,060–30,067Google Scholar
  10. 10.
    Aggeler, J, Fnsch, S in, and Werb, Z (1984) Collagenase is a maJor gene product of induced rabbit synovlal fibroblasts J Cell Bzol 98, 1656–1661CrossRefGoogle Scholar
  11. 11.
    Imal, T, FuJimaki, H, Abe, T, and Befus, D (1993) In vitro translation of mRNA from rat peritoneal and intestinal mucosal mast cells Int Arch Allergy ZmmunoE 102, 26–32CrossRefGoogle Scholar
  12. 12.
    Chandler, P in (1982) The use of single-stranded phage DNAs in hybrid arrest and release translation Anal Bzochem 127,9–16CrossRefGoogle Scholar
  13. 13.
    Roest, P A, Roberts, R G, van der Tuqn, A C, Helkoop, J C, van Ommen, G J, and den Dunnen, J T (1993) Protein truncation test (PTT) to rapidly screen the DMD gene for translation termmating mutations Neuromuscul Dlsord 3, 391–394CrossRefGoogle Scholar
  14. 14.
    Nicholls, P J, Johnson, V G, Blanford, M D, and Andrew, S in (1993) An Improved method for generating single-chain antlbodles from hybrldomas J Immunol Methods 165,81–91PubMedCrossRefGoogle Scholar
  15. 15.
    Curran, T, Gordon, M B, Rubmo, K L, and Sambucettl, L C (1987) Isolation and characterlzatlon of the c-fos(rat) cDNA and analysis of post-translational modlficatlon in vitroOncogene 2, 79–84PubMedGoogle Scholar
  16. 16.
    Mercuno, F, DlDonato, J, Rosette, C, and Karm, M (1993) ~105 and p98 percursor protems play an active role in NF-d-mediated signal transduction Genes Devel 7,705–718CrossRefGoogle Scholar
  17. 17.
    Walter, P and Blobel, G (1983) Preparation ofmlcrosomal membranes for cotranslatlonal protem translocatlon Methods Enzymol 96,84–93PubMedCrossRefGoogle Scholar
  18. 18.
    Tyrnms, M J and McInnes, B (1988) Efficient In Vztro expression of interferon CL analogs using SP6 polymerase and rabbit retlculocyte lysate Gene Anal Techn 5,9–15CrossRefGoogle Scholar
  19. 19.
    Johnston, J C and Rochon, D in (1990) Translation of cucumber necrosis virus RNA in vitroJ Gen Vzrol 71,2233–2241CrossRefGoogle Scholar
  20. 20.
    Brown, E A, ZaJac, A J, and Lemon, S in (1994) In vitro characterlzatlon of an internal rlbosomal entry site (IRES) present within the 5′ nontranslated region of hepatitis A virus RNA comparison with the IRES of encephalomyocardltls virusJ Vzrol 68,1066–1074Google Scholar
  21. 21.
    Clapp, L L and Patton, J T (1991) Rotavirus morphogenesls domains in the maJor inner capsld protem essential for bmding to angle-shelled particles and for trlmerlzatlon VzroZogy 180,697–708Google Scholar
  22. 22.
    Gerlinger, P, Mohler, E, Le Meur, M A, and Hlrth, L (1977) Monoclstromc translation of alfalfa mosaic virus RNAsNucleic Acids Res 4, 813–826PubMedCrossRefGoogle Scholar
  23. 23.
    Grubman, M J, Morgan, D O, Kendall, J, and Baxt, B (1985) Capsld intermediates assembled in a foot-and-mouth disease vn-us genome RNA-programmed cell-free translation system and in infected cells J Vwol 56, 120–126Google Scholar
  24. 24.
    Hardy, W R and Strauss, J H (1989) Processing the nonstructural polyprotems of smdbls vnus nonstructural protemase is in the C-terminal half of nsP2 and functions both in cls and in trans J Vwol 63,4653–4664Google Scholar
  25. 25.
    Pawson, T and Martin, G S (1980) Cell-free translation of avlan erythroblastosls virus RNAJ Vzrol 34,280–284Google Scholar
  26. 26.
    Demangeat, G, Hemmer, O, Frttsch, C, Le Gall, O, and Candresse, T (1991) In vitro processing of the RNA-2-encoded polyprotem of two nepovuuses tomato black ring vu-us and grapevme chrome mosaic virusJ Gen Vwoi 72,247–252CrossRefGoogle Scholar
  27. 27.
    Hellen, C U, Lm, Y Y, and Cooper, J I (1991) Synthesis and proteolytic processing of arabis mosaic nepovtrus, cherry leaf roll nepovtrus, and strawberry latent ringspot nepovirus proteins in reticulocyte lysate Arch Vwol 120, 19–31Google Scholar
  28. 28.
    Tymms, M J (1995) Quantitative measurement of mRNA using the RNase protection assay, in Methods En Molecular Bzology, vol 37 In Vztro Transcrlptlon and Translation Protocols(eiTymms, M J, ed), Humana, Totowa, NJ, pp 31–46Google Scholar
  29. 29.
    lack, M E and Loeb, L A (1993) Identttication of important residues wrthm the putative nucleostde bmding site of HSV-1 thymtdme kmase by random sequence selection analysts of selected mutants in vitro Bzochemzstry 32, 11,618-l1,626Google Scholar
  30. 30.
    Tobtas, K E and Kahana, C (1993) Intersubumt location of the active site of mammalian ormthme decarboxylase as determined by hybridization of sue-directed mutants BEOchemistry 32,5842–5847CrossRefGoogle Scholar
  31. 31.
    Howe, A Y, Elliott, J F, and Tyrrell, D L (1992) Duck hepatitis B vnus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcrtptase activitiesBzochem Bzophys Res Commun 189, 1170–1176CrossRefGoogle Scholar
  32. 32.
    Otterness, D in, Wteben, E D, Wood, T C, Watson, W. G, Madden, B J, McCormick, D. J, and Wemshilboum, R in (1992) Human liver dehydroepiandrosterone sulfotransferase molecular cloning and expression of cDNA Mol Pharmacol 41, 865–872PubMedGoogle Scholar
  33. 33.
    Sheffield, W P and BlaJchman, M A (1995) Deletton mutagenesis of heparm cofactorII defining the mmimum stze of a thrombm mhibiting serpmFEBS Lett 365, 189–192PubMedCrossRefGoogle Scholar
  34. 34.
    Denker, B in, Neer, E J, and Schmidt, C J (1992) Mutagenesis of the ammo termmus of the alpha subunit of the G protein Go In vitro characterization of alpha o beta gamma interactions J Blol Chem 267,6272–6277Google Scholar
  35. 35.
    Ramsay, R G (1995) DNA-binding studies using in vitro synthesized Myb proteins, in Methods wz Molecular Biology, vol 37 In Vitro Transcrlptlon and Translation Protocols (Tymms, M J, ed), Humana, Totowa, NJ, pp 369–378Google Scholar
  36. 36.
    Lim, F, Kraut, N, Frampton, J, and Graf, T (1992) DNA bmding by c-Ets-1, but not v-Ets,is repressed by an mtramolecular mechanism EMBO J 11,643–652PubMedGoogle Scholar
  37. 37.
    Lan, M S, Wasserfall, C, Maclaren, N K, and Notkms, A L (1996) IA-2, a transmembrane protein of the protein tyrosme phosphatase family, is a maJor autoantigen in msulm-dependent diabetes melhtusProc Natl Acad Scz USA 93,6367–6370CrossRefGoogle Scholar
  38. 38.
    Law, R H P and Nagley, P (1995) Import in isolated yeast mttochondria of radiolabelled proteins synthesized in vitro, in Methods In Molecular Biology vol 37 In Vitro Transcnptlon and Translatzon Protocols (Tymms, M J, ed), Humana, Totowa, NJ, pp 293–315CrossRefGoogle Scholar
  39. 39.
    Abbott, W in and Feizi, T (1991) Soluble 14-kDa beta-galactoside-specific bovine lectin Evidence from mutagenesis and proteolysis that almost the complete polypeptide chain is necessary for mtegrity of the carbohydrate recognmon domainJ BEOI Chem 266,5552–5557Google Scholar
  40. 40.
    Austin, R C, Rachubmski, R A, Fernandez-Rachubmski, F, and BlaJchman, M A (1990) Expression in a cell-free system of normal and variant forms of human anttthrombm III Ability to bmd heparm and react with alpha-thrombm Blood 76, 1521–1529PubMedGoogle Scholar
  41. 41.
    Dessens, J T. and Lomonossoff, G P (1991) Mutational analysts of the putative catalytic triad of the cowpea mosaic virus 24K protease Vzrology 184, 738–746CrossRefGoogle Scholar
  42. 42.
    Kumar, V, Green, S, Staub, A, and Chambon, P (1986) Localtsatton of the oestradtolbmding and putative DNA-binding domains of the human oestrogen receptor EMBO J 5,2231–2236PubMedGoogle Scholar
  43. 43.
    Marc, D, Drugeon, G, Haenm, A L, Gerard, M, and van der Werf, S (1989) Role of myrlstoylatlon of pol~ovu-us capsld protein VP4 as determined by site-dlrected mutagenesis of Its N-termmal sequence EMBU J 8,2661–2668Google Scholar
  44. 44.
    Solomon, T L, Solomon, L R, Gay, L S, and Rubenstem, P A (1988) Studies on the role of actm′s aspartlc acid 3 and aspartlc acid 11 using ohgodeoxynucleotlde-directed site-specific mutagenesis J BzoE Chem 263, 19,662-l 9,669Google Scholar
  45. 45.
    Wame, G J, Tymms, M J, Brandt, E R, Cheetham, B F, and Lmnane, A W (1992)Structure-tinctlon study of the region encompassing residues 2640 of human interferon-a4 ldentlficatlon of residues Important for antwlral and antlprohferatwe actlvltles J Interferon Res 12,43–48CrossRefGoogle Scholar

Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

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

Personalised recommendations