Volume 1

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

    60/668 (page 38)
    CHAPTER 2 Synthesis of the Polypeptide Chain Components of the Apparatus for Protein Synthesis Sequential Synthesis of Proteins The simplest way to interpret the genetic code is for amino acids to be added sequentially to the growing polypeptide chain as non-overlapping triplets are read from the starting point. (The alternative that all the amino acids are positioned on the template, after which peptide bonds are synthesised to connect them would reopen the question of how triplets are recognised only in the correct phase.) Dintzis (1961) and Naughton and Dintzis (1962) con firmed experimentally that the synthesis of haemoglobin in reticulocytes is sequential; their technique was to replace one of the amino acid precursors by an isotopie radioactive form during incubation. If amino acids are positioned before peptide bond synthesis, the label should be taken up equally into all parts of the protein. But if synthesis is sequential, the amino acids in those parts of the protein chains completed before the switch to radioactive conditions should be normal and only those parts of the chain synthesised after the substitution can contain the isotope. The point where radioactive replace normal amino acid is different for each chain syn thesised. However, if the radioactive content of completed haemoglobin molecules is analysed, only the far end should be labelled after a short time of incubation. Figure 2.1 shows that as incubation proceeds and more poly peptide chains are completed, the radioactive label moves towards the starting end. The procedure used in these experiments is to replace C 14 -leucine by H 3 - leucine in the incubation mixture and then to allow protein synthesis to continue for varying lengths of time before completed haemoglobin proteins are isolated and degraded to peptides by treatment with trypsin. When the peptides are fingerprinted by two dimensional chromatography, the levels of each radioactive isotope can be counted and the HMabel expressed relative to the C 14 -label, which acts as an internal control of the absolute level of leucine radioactivity—some peptides might have one or two leucine residues, others three or four.