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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024949-13
Application #
3272683
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1978-09-01
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1992-08-31
Support Year
13
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Gross, M; Rubino, M S; Hessefort, S M (1991) The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet). Biochem Biophys Res Commun 181:1500-7
Gross, M; Rubino, M S (1989) Regulation of eukaryotic initiation factor-2B activity by polyamines and amino acid starvation in rabbit reticulocyte lysate. J Biol Chem 264:21879-84
Wagner, T; Sigler, P B; Gross, M (1989) Mechanism of inhibition of eukaryotic translational initiation by the trinucleotide ApUpG. FEBS Lett 250:147-52
Gross, M; Rubino, M S; Starn, T K (1988) Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha. J Biol Chem 263:12486-92
Gross, M; Redman, R (1987) Effect of antibody to the hemin-controlled translational repressor in rabbit reticulocyte lysate. Biochim Biophys Acta 908:123-30
Gross, M; Wing, M; Rundquist, C et al. (1987) Evidence that phosphorylation of eIF-2(alpha) prevents the eIF-2B-mediated dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes. J Biol Chem 262:6899-907
Gross, M; Nguyen, T; Redman, R et al. (1986) Use of an antibody to characterize and determine the role of the major Met-tRNAf deacylase from rabbit reticulocyte ribosomes. Biochim Biophys Acta 867:220-8
Gross, M; Redman, R; Kaplansky, D A (1985) Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits. J Biol Chem 260:9491-500