mutant strains and the levels of b-galactosidase activities were measured in at least three independent experiments with three individual transformants in triplicates for each construct. These values were then expressed relative to the value obtained with the wt uORF1-GCN4-lacZ construct that was set to 100% in both strains. The mean values of the resulting percentages (with standard deviations) from all experiments were calculated and plotted (Figure 2B). We opted for this percentage expression because it enables a better comparison of the effects of the 59 sequences deletions on relative b-galactosidase activities indepen- dently in each strain. It is important to remember, however, that the a/tif32-D8 mutation itself reduces expression of the GCN4-lacZ from the uORF1-GCN4-lacZ constructs by ,70% when compared to wt a/TIF32 [10], and the chosen way of data presentation does not reflect this dramatic difference in activities. Owing to this ‘‘scaling up’’ we set a cut-off line of 80% for changes that are considered significant in the a/tif32-D8 mutant cells. (For comparison, the raw, not-normalized data for some of the constructs are shown in Figure S1A and S1B). It is also important to note that mRNAs produced from all GCN4-lacZ constructs used throughout the study are highly stable in both wt and a/tif32-D8 strains thanks to the fact that they all contain an intact stabilizer element (STE) that protects the natural GCN4 mRNA from NMD [10,20] (Figure S1C). As shown in Figure 2B, deletions of up to 16 nt from the 39 end of the 59 sequences (DEL6 and DEL16) did not produce any significant changes in the GCN4-lacZ expression in the wt cells. In contrast, larger deletions of 26, 36, and mainly of 46 nt (DEL 26, DEL36, and DEL46) reduced b-galactosidase activities by ,10%, ,40%, and ,60%, respectively. None of the largest deletions (DEL56 through DEL109) decreased the levels of GCN4-lacZ expression any further (i.e. above 60% of DEL46). In striking contrast to the wt cells, DEL36 had virtually no effect in the a/ tif32-D8 cells, whereas DEL46 led to a substantial drop in activity (by ,40%). None of the largest deletions decreased the GCN4-lacZ expression in a/tif32-D8 any further, just like in a/TIF32. Taken together, these results indicate the existence of two REI-promoting elements (RPE) falling between nt 231 and 261. The first element (RPE i.; 231 through 251) appears to function in the a/TIF32- NTD-dependent manner, since its removal in DEL36 shows genetic epistasis (non-additive phenotype) with the a/tif32-D8 mutation. The second REI-promoting element (RPE ii.; 251 through at least -61), however, operates independently of the a/ TIF32-NTD as its deletion together with the RPE i. in DEL46 produced a sharp decrease in b-galactosidase activities in both wt as well as mutant cells. Next we wanted to examine whether the far upstream sequence between nt in positions 2143 and 2181 constitutes yet another REI- promoting element of the 59 sequences as originally proposed by Grant and co-workers [14]. Towards this end, we deleted the corresponding region from the wt leader in DELup39 (Figure 2A) and observed ,25% and .30% reductions of activities in wt and a/ tif32-D8 mutant cells, respectively (Figure 2B). These results thus unambiguously reveal the presence of a third REI-promoting element (RPE iii.; 2143 through 2181) in the 59 sequences of uORF1 that seems to be less potent than the other two and that enhances the efficiency of REI in the a/TIF32-NTD-independent fashion. To conclude, our deletion analysis identified three RPEs that together make up what we designate the 59 enhancer of uORF1 thereafter. In silico prediction of the secondary structure of the 59 enhancer of uORF1 Having identified three RPEs in the 59 enhancer of uORF1, we wished to predict a potential secondary structure that the entire Translation Reinitiation Promoting Elements 220 nt long segment of the uORF1 59 UTR might progressively fold into during scanning for, translation elongation of, and termination on uORF1. Note that we excluded the most 39 terminal 9 nt from our analysis as they are highly likely buried in the mRNA binding channel of the 80S ribosome terminating at uORF1 [10]. The computer modeling was carried out by the RNA fold software [21]. Our prediction was based on two facts: 1) the 59 enhancer is not a standalone molecule with a rigid structure; its fold forms and changes dynamically as the sequence emerges from the ribosomal mRNA exit pore; and 2) the overall underrepresentation of Guanosines (the nucleotide composition of the entire 59 UTR of uORF1 is: A 40%, C 22%, G 7%, T 31%). Since the Gs are missing especially at the very 59 end of the sequence, we reasoned that their absence might leave this region unstructured, after it has emerged from the mRNA exit channel, owing to the fact that no local G–C pairs can be formed. To take these assumptions into account in our model, we divided the 59 UTR of uORF1 into three consecutive segments represented by the extreme 59 end 66-mer (AU-rich), the middle 81-mer, and the extreme 39 end 73-mer that is also AU-rich. We first folded the extreme 59 segment and found that, in agreement with our reasoning, the 66-mer showed no predictions of any secondary structures (Figure 2C). Importantly, it is believed that the AU-rich sequences have a stronger tendency to interact with proteins than those rich in Gs [22]. Hence it is conceivable that the extreme 59 AU-rich RNA stretch remains unstructured to engage in binding to ribosomal proteins and/or translation factors situated in the vicinity of the mRNA exit pore. Given this potential, we further stipulated that this 66-mer would not directly pair with the downstream sequences gradually leaving the exit channel during ribosomal scanning. To account for this, we added the middle 81- mer to the 66-mer and modeled the folding of the resulting 147- mer by blocking potential contacts between both individual segments. As a result, a short double-circle hairpin relatively GC-rich was predicted to form at the very 39 end of the 147-mer (Figure 2C). Interestingly, the same hairpin formed when the complete sequence of the 59 UTR of uORF1 was analyzed by RNA fold without any restraints (data not shown), and, furthermore, when homologues sequences from numerous yeast species were subjected to computer modeling (JP and LV, unpublished observations). These results indicate that the double-circle hairpin is a conserved structure, at least among various yeasts, that may have a functional significance in the translational control mechanism of GCN4 (see below). Finally, we added the remaining extreme 39 end segment to the pre-folded 147-mer and sought predictions of the overall structure of the 59 sequences. As shown in Figure 2C, the 73-mer remained mostly unfolded with the exception of a 9-nt long stem loop, situated only 6 nt downstream of the 39 end of the double-circle hairpin, with one 3-nt topical bulge and one 1-nt bulge close to its 39 end. Taken together with our genetic deletion analysis presented above, we propose that both the RPE i. and RPE iii. remain unstructured, whereas the RPE ii. folds into a stable stem loop with two bulges (Figure 2C). RNA structure probing of the 59 enhancer of uORF1 To test our computer predictions experimentally, we subjected a commercially synthesized 79-mer containing both the double- circle hairpin and the RPE ii. stem to enzymatic probing. (The 79- mer that was chosen based on RNA fold predictions starts 2 nt before the hairpin and ends 2 nt after the RPE ii. stem (Figure 3A).) The 79-mer was 59-end labelled by T4 polynucleotide kinase with [32P]-cATP, heated at 90uC for 3 minutes, slowly cooled down to room temperature to stimulate proper re-folding, and probed by PLoS Genetics | www.plosgenetics.org 5 July 2011 | Volume 7 | Issue 7 | e1002137