Figure 1.
REI-promoting activity of the 5′ sequences of uORF is strictly dependent on that of the 3′ enhancer.
(A) Schematic of the GCN4 mRNA leader showing distribution of all four short uORFs (REI-permissive uORF1 is labeled green; REI-non-permissive uORFs 2–4 are labeled red), the predicted structure of the uORF1's 5′ cis-acting sequences (5′ enhancer) defined in this study, 40S- and 80S-bound eIF3, and the description of the mechanism of the GCN4 translation control. The 3a and 4a “GCN4-expression repressed” steps take places under non-starvation conditions with abundant ternary complex (TC) levels, whereas the 3b and 4b “GCN4-expression derepressed” steps occur under starvation condition with limited supply of the TC (see text for further details). (B) Schematic showing predicted position of the 40S ribosome terminating at the stop codon of uORF1 from the GCN4 mRNA leader (adapted from [10]). E, P, and A sites of the 40S ribosome are aligned with the last two coding triplets and the TAA stop codon; entry and exit pores of the mRNA binding channel are labeled. The locations of the uORF1's 5′ sequences/enhancer (interacting with the NTD of a/TIF32), the 3′ enhancer (proposed to contact 18S rRNA), linker, and buried parts of the sequences upstream of uORF1 are indicated. The interaction between the a/TIF32-NTD and the small ribosomal protein RPS0A is depicted by a double headed arrow. (C) Schematic showing the GCN4-lacZ construct containing solitary uORF1, the surrounding sequences of which were divided into four separate segments (A1–D1; see text for further details). Arrows indicate replacements of these segments with the corresponding segments (A4–D4) surrounding uORF4, shown to the right of the arrows. (D) Various GCN4-lacZ constructs with the segment's combinations indicated in the first column were introduced into the YBS47 strain. The resulting transformants were pre-cultured in minimal media overnight, diluted to OD600 ∼0.35, grown for additional 6 hrs and the β-galactosidase activities were measured in the WCEs and expressed in units of nmol of o-nitrophenyl-b-D-galactopyranoside hydrolyzed per min per mg of protein. The mean values and standard deviations obtained from at least 3 independent measurements with three independent transformants, and activity in the mutant constructs relative to wt, respectively, are given in right column.
Figure 2.
The 5′ 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 5′ UTR defined below and used in panel B. (B) The YBS47 (a/TIF32) and YBS53 (a/tif32-Δ8) 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 Δ8 cut-off line. (C) In silico prediction of the secondary structure of the 5′ enhancer of uORF1 (nt −229 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.
Figure 3.
RNA structure probing of the 5′ enhancer of uORF1.
(A) In silico prediction of the secondary structure of the 79-nt segment of the 5′ 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 5′ enhancer segment comprising the RPEs ii. and iv. The latter 79-mer was 5′-end labeled with [γ-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.
Figure 4.
Identification of the forth REI-promoting element within the 5′ enhancer that acts in synergy with the RPE i. in the a/TIF32-NTD–dependent manner.
(A–C) Schematics showing the solitary uORF1 GCN4-lacZ construct with the battery of substitutions and/or deletions in the RPE i. (A), RPE ii. (B), and RPE iv. (C) that are used in the next three panels, respectively. (D) The RPE i. acts in the a/TIF32-NTD-dependent manner. The YBS47 and YBS53 strains were introduced with the GCN4-lacZ substitution constructs described in panel A and Figure 1D, and analyzed as in Figure 2B. wt, construct 1111; bg*, construct 4411. (E) The RPE ii. acts in the a/TIF32-NTD-independent manner. The strains as in panel D were introduced with the GCN4-lacZ deletion or substitution constructs described in panel B and analyzed as in Figure 2B. (F) The RPE iv. acts in synergy with the RPE i. in the a/TIF32-NTD-dependent manner. The strains as in panel D were introduced with the GCN4-lacZ deletion and/or substitution constructs described in panel C and analyzed as in Figure 2B.
Figure 5.
The extreme NTD of a/TIF32 contains two distal regions that promote efficient REI in the 5′ enhancer-dependent manner.
(A) Schematic representation of the first 200 amino acid residues of a/TIF32 shown as numbered circles (Boxes 1–20), each of them composed of 10 consecutive residues that were substituted with a stretch of 10 alanines. The sequence of Boxes 6, 8, and 17 is given below the schematic. (B) The a/tif32-Boxes 6, 8, and 17 impart a strong Gcn- phenotype. YBS52 (GCN2 a/tif32Δ) was transformed with individual YCplac111-based plasmids carrying the indicated a/TIF32 alleles and the resident YCpTIF32-His-U plasmid was evicted on 5-FOA. The resulting strains, together with isogenic strains H2880 (GCN2 a/TIF32; row 1) and H2881 (gcn2Δ a/TIF32; row 2), were then spotted in five serial 10-fold dilutions on SD (left panel) or SD containing 30 mM 3-AT (right panel) and incubated at 30°C for 3 and 6 days, respectively. (C) The failure of the a/tif32-NTD-Box mutations to derepress GCN4 is caused by a defect in resumption of scanning of post-termination 40S ribosomes on uORF1. Selected strains described in section B were introduced with the GCN4-lacZ constructs p180 (i.), pG67 (ii.), pM199 (iii.), and p209 (iv.), respectively, and analyzed as in Figure 2B. To induce the GCN4-lacZ expression (section i.), the transformants grown at the minimal media for 2 hrs after dilution were treated with 10 mM 3-AT for 6 hrs (a/TIF32) or overnight (Box mutants). An asterisk indicates data taken from [10] for comparison purposes. (D) Indicated strains described in section B were introduced with the GCN4-lacZ deletion constructs described in Figure 1D and Figure 4A–4C and analyzed as in Figure 2B. The RPEs affected by individual mutations are indicated above the bar diagram. wt, construct 1111; bg*, construct 4411.
Figure 6.
The 5′ sequences of the REI-permissive uORF of YAP1 contain structurally similar features to the RPEs of GCN4's uORF1 and promote REI in co-operation with the a/TIF32-NTD.
(A) In silico prediction of secondary structures of the 5′ sequences of uORF1 of GCN4 (nt −131 through −10), uORF of YAP1 (nt −81 through −1), and uORF1 of YAP2 (nt −101 through −4) carried out with the RNA fold software [21]. Pair wise structural similarities of 5′ sequences of GCN4 with 5′ sequences of YAP1 and YAP2 were computed using the RNA distance program [21]. Numbered nucleotides in the YAP1 sequence indicate mutated positions as illustrated in panel D. (B) Schematic showing the GCN4-lacZ construct containing solitary uORF1 (G4-uORF1), whose 5′ sequences past the trailing edge of the post-termination 40S ribosome (mRNA exit pore) were replaced by the corresponding 5’ sequences of either uORF of YAP1 (Y1-uORF1) or uORF1 of YAP2 (Y2-uORF1). YAP1 and YAP2 constructs where the individual genes were fused with lacZ while their 5′ UTRs were kept intact are also shown (Y1-lacZ and Y2-lacZ, respectively). (C) The YBS47 and YBS53 strains were introduced with the lacZ constructs described in panel B and analyzed as in Figure 2B. (D) The YBS47 and YBS53 strains were introduced with two structural mutants in the 5′ sequences of YAP1 (specified in panel A) inserted into Y1-uORF1 and analyzed as in Figure 2B.