Because of the importance of the types of hemoglobin synthesized during erythropoiesis to the clinical effects of sickle cell disease, we propose to continue our studies of the regulatory mechanisms that control hemoglobin synthesis at post- transcriptional levels. We will extend our studies of the translational control of globin synthesis by hemin in rabbit reticulocyte lysate by purifying the mediator of this effect, the hemin-controlled translational repressor (HCR), its precursor (ProHCR), and a soluble protein (supernatant factor) that neutralizes HCR and then prepare and examine the effect of antibodies to the purified proteins. We will test whether there is a change in exposed antigenic determinants in the transition from proHCR to HCR. We will study the activation and inactivation of HCR in the intact lysate and the modulating effects of hemin, the supernatant factor, a heat-stable, hexane extractable inhibitor from beef heart, and oxidized glutathione. We will determine the basis for the requirement for glucose-6-phosphate in translation that appears to be mediated at a level that is separate from the suppression of HCR activation. Finally, we plan to purify and characterize two additional enzymatic activities in rabbit reticulocyte; peptidyl-tRNA hydrolase and a 3' exonuclease. The former has not been isolated or characterized from higher organisms and may play an important role in translation. The 3'-exonuclease is an activity we have recently identified that degrades oligoribonucleotides to 5' mononucleotides and an ultimate dinucleotide monophosphate. It probably functions with RNAase in the phasing out of unneeded RNA during terminal erythroid differentiation.
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