Testing with puromycin for amino acyl trnas that limit the rate of peptide chain extension

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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 a hemoglobin chain has three isoleucyl residues and the β chain none. L-o-methylthreonine thus inhibits α globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting a 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 β chain synthesis in rabbit reticulocytes is more sensitive than α to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of β 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 γ and β chains remains constant.

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