Orf Uuu Ccg

Figure 17.5. Scheme of the yeast GCN4 mRNA. Four very short ORFs precedes the coding sequence for the GCN4. (Reproduced from A.G. Hinnebusch, R.C. Wek, T.E. Dever, A.M. Cigan, L. Feng & T.E. Donahue, in "Translational Regulation of Gene Expression 2", J. Ilan, ed., p.p. 87-115, Plenum Press, New York, 1993, with permission).

the inhibition of the initiation at the downstream GCN4 coding sequence. The ORFs inhibit the initiation at the downstream GCN4 start under normal conditions when nutrients are abundant. Under starvation conditions, however, the inhibition by the ORFs is reduced or abolished. The "derepression" of GCN4 correlates with the starvation-induced phosphorylation of eIF2. It has been found that the phosphorylation of eIF2 controls the effect of the ORFs on the efficiency of reinitiation at the GCN4 start. The first and the fourth ORFs seem to take the most important part in the regulation. The deletion of the first ORF reduces the GCN4 expression under derepressing (starvation) conditions and has no significant effect when the GCN4 translation is inhibited; this suggests the role of the first ORF as a positive control element. On the contrary, the deletion of the fourth ORF, or both the third and the fourth ORFs, abolishes the inhibition of the GCN4 initiation under repressing conditions, as expected if a negative control element is removed.

The model has been proposed (Fig. 17.6) that the reinitiation at the GCN4 start inversely depends on the level of the active (non-phosphorylated) eIF2. The ribosomes that bind to the 5'-end of the GCN4 mRNA will initiate at the first ORF, translate it and terminate. Under non-starvation conditions the active eIF2 is abundant, and so the terminated ribosomal particles will quickly reassociate with the eIF2:GTP:Met-tRNAi ternary complex. The part of them which remains to be bound with mRNA will reinitiate at the second ORF, and after termination again only a part will remain mRNA-bound and reinitiate, and so on. Thus, most of the ribosomes will be lost during termination and reinitiation at the four ORFs and, therefore, fail to reach the GCN4 start. Under starvation conditions, the level of the eIF2:GTP:Met-tRNAi ternary complex is reduced due to phosphorylation of eIF2. From this it may follow that the ribosomal particles after termination at the first ORF will scan the downstream sequence but will not have time to catch the ternary complex and reinitiate at the second, the third and the fourth ORFs. As a result, most of them may reach the initiation codon of the GCN4 coding sequence. According to the model,

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