circle hairpin owing to the fact that the other strand is too close to the 59 end label (nt 3–7). Since all four G’s that are distal to the 59- radiolabel (namely G23, G31, G48, and G75) were predicted to occur in the based paired regions, no cleavages with RNAse T1 (cleaves at 39 end of single-strand G’s) should be detected. The fact that we did reproducibly observe cuts at all four G’s (lanes T1) suggests that the 79-mer is metastable, undergoing dynamic unfolding/folding cycles in our sample. This is expected, however, given that the REI process requires the ribosome to smoothly scan through this region before it translates uORF1, stops at its stop codon and primes itself for resumption of scanning. It is understood that under given circumstances a highly stable secondary structure would actually impede swift translational remodeling of this critical region. Indeed, a critical support for the proposed structure identity was provided by the T1 enzyme under denaturing conditions (lane T1 denatur) that showed a substan- tially stronger T1 cuts compared to the folded sample (lane T1 fold). Identification of the fourth REI-promoting element within the 59 enhancer of uORF1 that acts in synergy with the RPE i. in the a/TIF32-NTD–dependent manner Next we subjected individual RPEs to an in-depth analysis in order to provide additional support for their importance in the REI mechanism of GCN4. The RPE i. acts in the a/TIF32-NTD- dependent manner and appears to be unstructured. Hence it is highly likely that the putative direct interaction between the a/ TIF32-NTD and the RPE i. is sequence specific. To test that, we divided the RPE i. into three consecutive segments with the first two comprising 9 nt (231 through 239 in SUB31; and 240 through 248 in SUB40), and the third one being composed of 6 nt (249 through 254 in SUB49) and ending at the base of the RPE ii stem (Figure 4A and Figure 2C). We then substituted sequences of these segments with complementary nt and tested the resulting constructs for efficiency of GCN4-lacZ expression. As shown in Figure 4D, whereas neither of the substitutions significantly affected expression in the a/tif32-D8 cells, SUB31 produced ,25%, and SUB40 and SUB49 even ,40% reductions, respectively, in wt cells. Hence the results obtained especially with the latter two substitutions nicely correlate with DEL36 that removes the entire element (Figure 2B and Figure 4F) and suggest that mainly the nature of nt situated at the 59 end of the RPE i. is critical for its function in REI. The RPE ii. forms a stem with two bulges and does not seem to be involved in the functional interaction of the 59 enhancer with the a/TIF32-NTD. We designed two constructs one of which removed all stem-forming nt and the other one replaced them with a stretch of multiple CAA triplets, which minimizes formation of secondary structures [23] (Figure 4B). As predicted, both constructs reduced the GCN4-lacZ expression by ,40% in wt as well as in a/tif32-D8 cells clearly confirming the importance of this element for resumption of scanning after uORF1 in the a/TIF32- NTD-independent fashion. We also swapped both strands of the stem either preserving the sequences of both bulges or replacing them with complementary nt to find out whether the structure or sequence, or both is important. In either case the GCN4-lacZ expression went down by consistent ,40% in both strains (data not shown), suggesting that certainly the sequence is critical for function of this element. The question of the fold importance could not be satisfactorily answered. As shown in Figure 2B, removal of the RPEs i. and ii. in DEL46 (216 through 261) produced ,60% drop in the b-galactosidase activity in wt cells and any of the larger deletions up to 2125 nt that we tested did not make it any worse. These findings may Translation Reinitiation Promoting Elements indicate that a nucleotide sequence from the 59 base of the RPE ii. stem (nt 276) upstream (at least up to nt 2125) is dispensable for the 59 enhancer function in REI. Interestingly, however, our computer modeling suggested that a nucleotide stretch spanning nt 2129 through 283 folds into the conserved double-circle hairpin (Figure 2C) that, by definition, would be expected to be functionally important. To test that, we employed computer modeling and designed a triple nucleotide substitution (C-129A, G-128A, G-109C) that should completely disrupt base-pairing between nt forming both stems while preserving the length and the rest of the sequence of this rather long segment intact (Figure 4C). As shown in Figure 4F, the resulting AA-C construct indeed reduced the GCN4-lacZ expression by ,40% but only in the wt cells. In principle, it behaved the same as the RPE i.-deletion construct DEL36 indicating that the RPE i. and this hairpin may closely cooperate with each other and also with the NTD of a/ TIF32. If true, then combining DEL36 and AA-C mutations (Figure 4C) should be epistatic; and this was exactly observed (Figure 4F). These findings thus identify a fourth REI-promoting element (RPE iv; 2129 through 283) within the 59 enhancer that adopts a conserved higher-order structure and acts in synergy with the RPE i. and the a/TIF32-NTD. The RPEs i., ii., and iv. of the 59 enhancer are critically required for up-regulation of GCN4 expression under starvation conditions All experiments described so far were carried out with GCN4- lacZ constructs carrying only uORF1 of the four uORFs from the GCN4 mRNA leader and under non-starvation conditions. To perform an ultimate test of our findings, we examined effects of selected mutations on GCN4 induction in wt cells treated with 3- aminotriazole (3-AT; an inhibitor of histidine biosynthetic genes that mimics starvation conditions) using a construct containing uORF1 and uORF4 that together suffice for wt regulation of GCN4 expression (Figure S2). As described in detail in the Text S1, obtained results underpinned the functional importance of all three major 59 enhancer’s RPEs (i., ii., and iv.) in their task to ensure efficient REI on GCN4 when cells are starved for nutrients such as amino acids. The extreme NTD of a/TIF32 contains two distal regions that promote efficient REI in the 59 enhancer-dependent manner The a/tif32-D8 mutation was shown to reduce the REI efficiency by two distinct mechanisms: (i) decreasing retention of eIF3 on elongating ribosomes translating uORF1 by reducing the binding affinity of eIF3 to 40S subunits and (ii) impairing functional interaction of a/TIF32 with the 59 enhancer of uORF1 [10]. To identify residues in the extreme NTD of a/TIF32 that are responsible for these two roles, and to possibly separate them, we introduced Ala substitutions in consecutive blocks of 10 residues between amino acids 1 and 200 (dubbed Boxes 1 to 20, Figure 5A). None of these mutations was lethal and only Boxes 6 (residues 51– 60), 8 (71–80), and 17 (161–170) produced slow-growth (Slg2) phenotypes and, most importantly, significant Gcn2 phenotypes (Figure 5B) indicating an impairment of the GCN4 induction. Indeed, our GCN4-lacZ reporter assays with the wt GCN4-leader confirmed the derepression defect (Figure 5C, construct i). Interestingly, combining Boxes 6+17 and 8+17 but not 6+8 exacerbated both the Slg2 and Gcn2 phenotypes of the single mutants (Figure 5B and 5C, construct i) suggesting the presence of two functionally partially redundant regions within the a/TIF32- NTD, with the first one represented by Boxes 6 and 8, and the PLoS Genetics | www.plosgenetics.org 8 July 2011 | Volume 7 | Issue 7 | e1002137