Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E⅐4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70S6k , resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucineinduced effects. Thus, leucine acting through an mTORdependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.Certain amino acids, notably the essential amino acids, not only serve as precursors for protein synthesis, but also have important regulatory roles in the initiation phase of mRNA translation (1-3). Regulation of translation initiation is known to occur through modulation of two of the numerous steps in the pathway. The first regulated step is the binding of methionyl-tRNA i (Met-tRNA i ) to the 40 S ribosomal subunit to form the 43 S preinitiation complex (reviewed in Refs. 4 and 5). This step is mediated by eukaryotic initiation factor, eIF2, and involves formation of an eIF2⅐GTP⅐Met-tRNA i ternary complex followed by binding of the ternary complex to the 40 S ribosomal subunit. The overall process of Met-tRNA i binding is regulated through changes in the activity of the guanine nucleotide exchange factor for eIF2, termed eIF2B, and appears to involve changes in phosphorylation of either the ␣-subunit of eIF2 and/or the ⑀-subunit of eIF2B.The second regulated step in translation initiation is the binding of mRNA to the 43 S preinitiation complex (reviewed in Refs. 4 and 5). This step is mediated by a group of proteins collectively referred to as eIF4. During this step, eIF4E binds to the m 7 GTP cap structure present at the 5Ј-end of essentially all eukaryotic mRNAs and, through association with eIF4G, also binds to the 40 S ribosomal subunit. The mRNA binding step is regulated through changes in phosphorylation of eIF4E, with phosphorylation increasing the affinity of eIF4E for the cap structure (6) as well as by changes in the availability of eIF4E to form the active eIF4E⅐eIF4G c...

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S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the pathway of polyamine biosynthesis. The cellular levels of the polyamines specifically regulate AdoMetDC translation through the 5-leader of the mRNA, which contains a small upstream open reading frame (uORF) 14 nucleotides from the cap. Mutating the initiation codon of the uORF, which encodes a peptide product with the sequence MAGDIS, abolished regulation. In addition, the uORF is sufficient, by itself, to provide polyamine regulation when inserted into the 5-leader of the human growth hormone mRNA. Changing the amino acid sequence at the carboxyl terminus of the peptide product of the uORF abolished polyamine regulation. In contrast, altering the nucleotide sequence of the uORF at degenerate positions, without changing the amino acid sequence of the peptide, did not affect regulation. Extending the distance between cap and uORF, thereby changing the rate of initiation at the initiator AUG of the uORF, did not alter polyamine regulation. When the uORF was extended so as to overlap, out of frame, the downstream major cistron, polyamine regulation was abolished. We propose that polyamines do not modulate the rate of recognition of the uORF but rather regulate interaction of the peptide product of the uORF with its target.