Translation Reinitiation Promoting Elements Figure 2. The 59 sequences of uORF1 contain at least three REI-promoting elements (RPEs), one of which operates in the a/TIF32- NTD–dependent manner. (A) Schematic showing the solitary uORF1 GCN4-lacZ construct with the battery of deletions in the uORF1’s 59 UTR defined below and used in panel B. (B) The YBS47 (a/TIF32) and YBS53 (a/tif32-D8) strains were introduced with the GCN4-lacZ deletion constructs described in panel A and Figure 1D and analyzed as in Figure 1D, except that YBS53 was grown for 8 hours. Arrows indicate constructs defining the individual RPEs; please see corresponding text for the definition of the D8 cut-off line. (C) In silico prediction of the secondary structure of the 59 enhancer of uORF1 (nt 2229 through -10) carried out with the RNA fold software [21]. Four individual RPEs identified in panel B and Figure 4 are labeled and color-coded. Division into three segments used for computer modeling is indicated. doi:10.1371/journal.pgen.1002137.g002 RNases T1 and V1 prior to analysis on denaturing polyacrylamide gels. As shown in Figure 3B, the data for enzymatic probing were in good agreement with the computationally predicted secondary structure of this 59 enhancer section. Formation of all three stems, two of which occur in the double-circle hairpin [nt 3–7 base- paired with nt 45–49; and nt 22–24 base-paired with nt 31–33], and the third forms the RPE ii [nt 56–64 base-paired with nt 68– 77], was confirmed by specific cleavages by RNase V1 (cuts based- paired nucleotides only; lane V1). As expected, V1 cuts of the RPE ii. stem are preferentially detected in the strand that is more proximal to the 59-radiolabel. On the other hand, V1 cuts are only detected in the more distal strand of the longer stem of the double- Figure 3. RNA structure probing of the 59 enhancer of uORF1. (A) In silico prediction of the secondary structure of the 79-nt segment of the 59 enhancer of uORF1 that was subjected to enzymatic probing. Scissors and light blue residues indicate cleavage sites of T1 and V1 RNases shown in panel B, respectively. (B) RNA structure probing of the commercially synthesized uORF1’s 59 enhancer segment comprising the RPEs ii. and iv. The latter 79-mer was 59-end labeled with [c-32P]-ATP and subjected to limited RNase cleavage using RNases T1 and V1 under denaturing (denatur) or folding-promoting (fold) conditions. Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (AH) and by the position of known RNase T1 cuts, determined empirically. Predicted double-stranded regions are indicated on the right- hand side of the panel and the shorter exposition of the upper portion of the gel showing T1 cuts is shown at the bottom of the panel. doi:10.1371/journal.pgen.1002137.g003 PLoS Genetics | www.plosgenetics.org 7 July 2011 | Volume 7 | Issue 7 | e1002137