REQUIREMENTS FOR INTERCISTRONIC DISTANCE AND LEVEL OF EUKARYOTIC INITIATION FACTOR-II ACTIVITY IN REINITIATION ON GCN4 MESSENGER-RNA VARY WITH THE DOWNSTREAM CISTRON

Translational control of the GCN4 gene in response to amino acid avail ability is mediated by four short open reading frames in the GCN4 mRNA leader (uORFs) and by phosphorylation of eukaryotic initiation factor 2 (eIF-2). We have proposed that reducing eIF-2 activity by phosphory lation of its alpha subunit or by a mutation in the eIF-2 recycling fa ctor eIF-2B allows ribosomes which have translated the 5'-proximal uOP F1 to bypass uORF2 to uORF4 and reinitiate at GCN4 instead. In this re port, we present two lines of evidence that all ribosomes which synthe size GCN4 have previously translated uORF1, resumed scanning, and rein itiated at the GCN4 start site. First, GCN4 expression was abolished w hen uORF1 was elongated to make it overlap the beginning of the GCN4 c oding region. Second, GCN4 expression was reduced as uORF1 was moved p rogressively closer to GCN4, decreasing to only 5% of the level seen i n the absence of all uORFs when only 32 nucleotides separated uORF1 fr om GCN4. We additionally found that inserting small synthetic uORFs be tween uORF4 and GCN4 inhibited GCN4 expression under derepressing cond itions, confirming the idea that reinitiation at GCN4 under conditions of diminished eIF-2 activity is proportional to the distance of the r einitiation site downstream from uORF1. While uORF4 and GCN4 appear to be equally effective at capturing ribosomes scanning downstream from the 5' cap of mRNA, these two ORFs differ greatly in their ability to capture reinitiating ribosomes scanning from uORF1. When the active fo rm of eIF-2 is present at high levels, reinitiation appears to be much more efficient at uORF4 than at GCN4 when each is located very close to uORF1. Under conditions of reduced recycling of eIF-2, reinitiation at uORF4 is substantially suppressed, which allows ribosomes to reach the GCN4 start site; in contrast, reinitiation at GCN4 in constructs lacking uORF4 is unaffected by decreasing the level of eIF-2 activity. This last finding raises the possibility that time-dependent binding to ribosomes of a second factor besides the eIF-2-GTP-Met-tRNA(i)Met t ernary complex is rate limiting for reinitiation at GCN4. Moreover, ou r results show that the efficiency of translational reinitiation can b e strongly influenced by the nature of the downstream cistron as well as the intercistronic distance.