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Testing with puromycin and amino acyl tRNAs that limit the rate of peptide chain extension
Abstract:
With puromycin one can recognize when the synthesis of a given protein is dependent on amino acyl tRNA that is present in rate limiting amount. We demonstrate this use of puromycin by its interaction with another inhibitor, L-o-methylthreonine. L-o-methylthreonine lowers the Ile-tRNA concentration in the cell, thereby inhibiting synthesis of proteins containing isoleucine. In certain rabbits, the alpha hemoglobin chain has three isoleucyl residues and the beta chain none. L-o-methylthreonine thus inhibits alpha globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting alpha globin synthesis. Hence, puromycin is a more effective inhibitor when the Ile-tRNA concentration is lowered. Cycloheximide and sodium fluoride have different modes of action from puromycin. Neither synergizes with L-o-methylthreonine; instead, the interaction is less than additive. We have found that beta chain synthesis in rabbit reticulocytes is more sensitive than alpha to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of beta chain elongation. The switch from fetal to adult hemoglobin in humans does not involve changes in limiting amino acyl tRNA because, for cord blood from infants of different developmental ages, the puromycin sensitivity of incorporation into gamma and beta chains remains constant.
Insights
Puromycin helps identify rate-limiting amino acyl-tRNAs in protein synthesis. It synergizes with L-o-methylthreonine, which lowers isoleucine-tRNA, enhancing inhibition of alpha-globin synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Puromycin is a tool to assess protein synthesis dependency on amino acyl-tRNAs.
- L-o-methylthreonine specifically reduces intracellular isoleucyl-tRNA levels.
- Alpha-globin chains in certain rabbits contain isoleucine, while beta-globin chains do not.
Purpose of the Study:
- To investigate the synergistic effects of puromycin and L-o-methylthreonine on protein synthesis.
- To explore the role of amino acyl-tRNA availability in regulating globin chain synthesis.
- To compare the sensitivity of alpha- and beta-globin synthesis to puromycin inhibition.
Main Methods:
- Utilizing puromycin as an inhibitor to probe tRNA-dependent protein synthesis.
- Employing L-o-methylthreonine to modulate isoleucyl-tRNA levels in rabbit reticulocytes.
- Assessing the impact of combined inhibitors on alpha- and beta-globin synthesis.
Main Results:
- Puromycin and L-o-methylthreonine exhibit synergistic inhibition of alpha-globin synthesis, indicating puromycin's enhanced efficacy when isoleucyl-tRNA is limited.
- Cycloheximide and sodium fluoride do not synergize with L-o-methylthreonine, suggesting different mechanisms of action.
- Beta-globin synthesis is more sensitive to puromycin inhibition than alpha-globin synthesis in rabbit reticulocytes.
- Human fetal to adult hemoglobin switching does not involve changes in limiting amino acyl-tRNAs, as puromycin sensitivity remains constant.
Conclusions:
- Puromycin's effectiveness as an inhibitor is modulated by the availability of specific amino acyl-tRNAs.
- Differential sensitivity of globin chains to puromycin may reflect sequence or tRNA-dependent elongation differences.
- Amino acyl-tRNA levels are not limiting factors in the human fetal to adult hemoglobin switch.