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STUDIES ON THE ROLE OF THE PROTEIN FACTOR RF IN THE REGULATION OF PROTEIN SYNTHESIS INITIATION IN RABBIT RETICULOCYTES (RESCUE LYSATE, HEME)
Abstract
During heme-deficiency in reticulocyte lysates, a latent translational inhibitor, named HRI (heme-regulated inhibitor), which blocks polypeptide chain initiation, is activated. HRI is a protein kinase that specifically phosphorylates the M(,r) 38,000 subunit of the Met-tRNA(,f) binding factor eIF-2. A previously reported eIF-2-ancillary protein factor Co-eIF-2 promotes displacement of GDP from the native eIF-2(.)GDP complex facilitating initiation complex (Met-tRNA(,f)(.)eIF-2(.)GTP) formation in the presence of physiological magnesium. Phosphorylated eIF-2 (eIF- 2(alpha)(P)) inhibits initiation complex formation as Co-eIF-2 does not displace GDP from eIF-2(alpha)(P)(.)GDP. RF, a high molecular weight lysate supernatant factor, reverses protein synthesis in heme-deficient lysates and also reverses HRI inhibition of initiation complex formation; RF contains Co-eIF-2- activity. RF also contains excess M(,r) 38,000 subunit of eIF-2 in the free and unphosphorylated form. The excess M(,r) 38,000 subunit of RF can only be phosphorylated by HRI and ATP in the presence of GDP. RF which promotes initiation complex formation with eIF-2(.){('3)H}GDP and accompanying GDP displacement can also promote initiation complex formation in the presence of HRI and ATP without accompanying GDP displacement. In the presence of HRI and ATP, the initiation complex formed using RF is active in AUG-codon directed Met-tRNA(,f)(.)40S formation; Met-tRNA(,f)(.)40S formation is inhibited by preincubation of RF with GDP. These activities are sensitive to N-ethyl maleimide. Further fractionation of RF yields a preparation which contains HRI-sensitive Co-eIF-2 activity and does not efficiently reverse protein synthesis initiation in heme-deficient reticulocyte lysates. This preparation does not contain excess M(,r) 38,000 subunit. These observations suggest that (1) RF provides unphosphorylated M(,r) 38,000 subunit to eIF-2(alpha)(P)(.)GDP and restores eIF-2 activity. (2) RF contains Co-eIF-2 activity which has dual functions: (i) stimulation of initiation complex by eIF-2 and (ii) GDP-displacement from eIF-2(.)GDP during initiation complex formation.
Subject Area
Biology
Recommended Citation
GRACE, MICHAEL JUDD, "STUDIES ON THE ROLE OF THE PROTEIN FACTOR RF IN THE REGULATION OF PROTEIN SYNTHESIS INITIATION IN RABBIT RETICULOCYTES (RESCUE LYSATE, HEME)" (1984). ETD collection for University of Nebraska-Lincoln. AAI8423786.
https://digitalcommons.unl.edu/dissertations/AAI8423786