We propose to continue and extend our recent observations of the effect of phosphorylation of the Alpha subunit of eukaryotic initiation factor-2 (eIF-2Alpha) in the unfractionated rabbit reticulocyte lysate, which represents the mechanism by which protein synthesis becomes suppressed in heme-deficiency or upon the addition of a low level of double-stranded RNA. Recent studies by other laboratories have indicated that when eIF-2Alpha becomes phosphorylated, the ability of a separate factor (termed RF) to promote the exchange of GTP for GDP bound to eIF-2 and thus its recycling is impaired. Our recent results suggest that inhibition is also due to the accumulation of 60S.eIF-2.GDP complexes and 48S complexes, with the secondary, enzymatic deacylation of 40S subunit-bound Met-tRNA-f. We will use antibody we have recently prepared to Met-tRNA-f deacylase, which totally inactivates enzyme activity, to determine the precise role the deacylase plays in the regulation of polypeptide chain initiation by phosphorylation of eIF-2Alpha. We will test in several ways our working hypothesis that eIF-2 and GTP or GDP normally recycle on 60S ribosomal subunits and may thus promte subunit joining and th at the phosphorylation of eIF-2alpha inhibits by preventing the dissociation of eIF-2 GDP from 60S subunits, which must be mediated by RF. We will examine the binding to ribosomal components of such radiolabeled probes as (14C)RF, (alpha-32P)GTP, (35S)Met-tRNA-f, and (3 H-guanyly) beta,gamma-imido-GTP under a variety of conditions in the intact lysate and with ribosomes isolated from lysate by chromatography on Sepharose 6B. We will determine the effect of antibodies, we are currently preparing against RF and eIF-2, on the ribosome binding of these radiolabeled probes under varied conditions. These antibodies will also be used to quantitate the endogenous eIF-2 and RF on ribosomal particles and in soluble complexes in lysate in the presence or absence of phosphorylation of eIF-2alpha. We will determine the activity and fate of gradient purified,k 40S, 60S, and 80S-associated initiation complexes from incunated lysate by reincubation. Finally, we will try to determine why protein synthesis with reconstituted, 0.5M K C1-washed ribosomes is insensitive to inhibition oby phosphorylation of eIF-2Alpha and whether this may be related to RF-mediated dissociation of eIF-2 GDP from 60S subunits.
