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Phosphorylation of initiation factor eIF-2 alpha, binding of mRNA to 48 S complexes, and its reutilization in
Abstract:
The formation of 80 S initiation complexes containing labeled viral mRNA was drastically inhibited when mRNA binding assays were carried out with reticulocyte lysate preincubated with double-stranded RNA (dsRNA). When the assays were analyzed by centrifugation on sucrose gradients, the mRNA incubated with lysate pretreated with dsRNA sedimented as a 48 S complex. Met-tRNA, GDP, and phosphorylated initiation factor eIF-2(alpha P) were shown to co-sediment with the 48 S complex. Therefore, the formation of this complex was attributed to the phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase. These observations suggested that mRNA could bind to a 40 S ribosomal subunit containing Met-tRNAf, GDP, and eIF-2(alpha P), but the joining of a 60 S ribosomal subunit was inhibited. When the 48 S complex was isolated and incubated with lysate without added dsRNA, the mRNA could form 80 S initiation complexes. The shift of mRNA from 48 S to 80 S complexes was also observed when the eIF-2 alpha kinase activity was inhibited by the addition of 2-aminopurine. This shift was quite slow, however, when compared to the rate of binding of free mRNA to 80 S initiation complexes. The 2-aminopurine was effective in reversing the inhibition of protein synthesis by dsRNA and in maintaining a linear rate of protein synthesis for 3 h in lysates. Without added 2-aminopurine, protein synthesis was inhibited after 90 min even in lysates supplemented with hemin and eIF-2(alpha P) was detected in these lysates. This finding indicated that eIF-2 alpha phosphorylation could be in part responsible for limiting the duration of protein synthesis in mammalian cell-free systems.
Insights
Double-stranded RNA (dsRNA) inhibits protein synthesis by activating a kinase that phosphorylates eIF-2 alpha, preventing 80 S initiation complex formation. This inhibition can be reversed by 2-aminopurine, restoring protein synthesis.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Protein synthesis initiation is a complex process involving multiple factors and ribosomal subunits.
- Double-stranded RNA (dsRNA) is known to trigger cellular defense mechanisms, including the inhibition of protein synthesis.
Purpose of the Study:
- To investigate the mechanism by which dsRNA inhibits protein synthesis initiation.
- To identify the specific molecular events leading to the observed inhibition.
Main Methods:
- Reticulocyte lysate was preincubated with dsRNA and subjected to mRNA binding assays.
- Sucrose gradient centrifugation was used to analyze the sedimentation of mRNA-containing complexes.
- The role of initiation factor eIF-2 alpha phosphorylation was examined.
Main Results:
- dsRNA preincubation led to the formation of 48 S initiation complexes instead of the usual 80 S complexes.
- These 48 S complexes contained Met-tRNA, GDP, and phosphorylated eIF-2 alpha (eIF-2(alpha P)).
- The phosphorylation of eIF-2 alpha by a dsRNA-activated protein kinase was identified as the cause of inhibition.
- Inhibition was reversed by 2-aminopurine, which also restored protein synthesis.
Conclusions:
- dsRNA inhibits protein synthesis by promoting eIF-2 alpha phosphorylation, which blocks the joining of ribosomal subunits.
- eIF-2 alpha phosphorylation is a key factor limiting protein synthesis duration in mammalian cell-free systems.
- 2-aminopurine can overcome dsRNA-induced inhibition and prolong protein synthesis.