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The nature of the genet in rode, once determined, ted to further questions about how a polynucleotide chain directs the synthesis of a polypeptide. As we have seen here and shall discuss in more detail in Chapter 6, polynucleotide chains (both DNA and RNAJ are synthesized in a 5J 3' direction. But what about the growing polypeptide chain? Is it assembled in an amino-terminal to carboxyl-terminal direction, or the opposite?

This question was answered in a classic experiment in which a cell-free system was used for carrying out protein synthesis. The cell-free system was created using an extract from immature red blood cells (known as reticulocytes) from a rabbit, which are efficient factories for the synthesis of the i*- and p-globin subunits of hemoglobin. The cell-free system was treated with a radioactive amino acid for a very few seconds (/ess than the time required to synthesize a complete glohm chain) after wrhich protein synthesis was immediately stopped. A brief radioactive labeling regime of this kind is known as a pulse or pulse-labeling. Next, globin chains that had completed their growth during the period of the pulse-labeling were separated from incomplete chains by gel electrophoresis (Chapter 20). The full-length polypeptides were then treated with an enzyme, the protease trypsin, that cleaves proteins on particular sites in the polypetide chain, thereby generating a series of peptide fragements. In the final step of the experiment, the amount of radioactivity that had been incorporated into each peptide fragment was measured (Figure 2-19).

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