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Initiation of Globin Synthesis

PREPARATION AND USE OF RETICULOCYTE RIBOSOMES RETAINING INITIATION REGION MESSENGER RIBONUCLEIC ACID FRAGMENTS

From the ¹ From the Section on Molecular Hematology, National Heart and Lung Institute, Bethesda, Maryland 20014

Rabbit reticulocyte lysate ribosomes, exposed briefly to pancreatic RNAse and then treated with 0.5 m KCl and puromycin, are converted to ribosomes that carry fragments of globin and ß messenger RNA. These ribosomes support several initiation reactions in the absence of added mRNA including: (a) binding of the initiator tRNA, Met-tRNA_f, to the small ribosomal subunit; (b) synthesis of the naturally occurring initial dipeptide, Met_f-valine; (c) synthesis of the initial tripeptides for the and ß chains, Met_f-valyl-leucine (a) and Met_f-valyl-histidine (ß). All of these reactions require protein initiation factors, the initiator tRNA, a low Mg⁺⁺ concentration and GTP. Complete and ß globin chains can be synthesized only when exogenous globin mRNA is added to the system. Thus, lysate ribosomes treated with low levels of RNAse have almost no intact endogenous globin mRNA, but they still retain a significant number of mRNA fragments which contain the initiation region.

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