Translation Reinitiation Promoting Elements Figure 1. REI-promoting activity of the 59 sequences of uORF is strictly dependent on that of the 39 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 59 cis-acting sequences (59 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 59 sequences/enhancer (interacting with the NTD of a/TIF32), the 39 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 b-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. doi:10.1371/journal.pgen.1002137.g001 uORF in order to facilitate efficient resumption of scanning of the 40S ribosomal subunit. Results The 59 and 39 sequences of uORF1 closely cooperate in stimulating efficient REI A considerable difference in efficiency of resumption of scanning following translation of uORF1 versus uORF4 in the GCN4 mRNA leader is thought to be attributable to the distinct sequences surrounding the termination codons of these two uORFs. Replacing the last codon and 10 nt downstream of the uORF1 stop codon (Figure 1B, dubbed the 39 enhancer) with the corresponding nucleotides from uORF4 was sufficient to make uORF1 as inhibitory for REI on GCN4 as is uORF4 [13]. Similarly, sequences located in the leader region .20 nt upstream of the AUG start codon of uORF1 (Figure 1B) were also shown to be critically required for efficient REI downstream [14]. However, individual contributions of both of these stimulatory sequences to the overall REI efficiency have never been directly compared in a single experiment. To do that, we divided the two uORFs and their surrounding sequences into four segments: segment A (166 bp in length from position -181 to -16 relative to the AUG start codon corresponding to the 59 REI-promoting sequences of uORF1); segment B (15-bp long segment (215 to 21) designated previously as linker [10]); segment C (3 coding triplets and a termination codon); and segment D (25 bp downstream from the uORF stop codon including the aforementioned 39 enhancer of uORF1) (Figure 1C). It should be noted that the A segment of uORF4 has the start codons of the preceding uORFs 2 and 3 mutated out to compare the effects of only uORFs 1 and 4. Also, in contrast to A, C and D segments, the sequence corresponding to the B-linker region of uORF1 was previously shown to play a negligible role for efficient REI [19]. Three hybrid uORFs were constructed by the substitution of some or all of uORF1 segments with the corresponding segments derived from uORF4 in the GCN4-lacZ construct lacking all three uORFs naturally occurring downstream of uORF1 (compare Figure 1A and 1C). When all four uORF1 segments were replaced by the corresponding uORF4 segments (Figure 1D; row 2 (construct 4-4-4-4)), the GCN4-lacZ expression dropped by ,20-fold to the background level (Figure 1D, row 2 [bg] versus 1 [wt]) in accord with previous findings demonstrating the two uORFs’ highly disparate capacities to promote efficient REI [19]. Selective replacements of either the 59 sequences or the entire 39 enhancer (row 4 (construct 4-4-1-1) versus row 3 (1-1-4-4)) of uORF1 resulted in 6-fold or 17-fold reductions in b-galactosidase activities, respectively. These data indicate that both elements closely co-operate to promote highly effective REI downstream of uORF1, but probably by mechanis- tically distinct processes. Interestingly, whereas the 39 enhancer is sufficient to stimulate resumption of scanning to at least some degree (by ,13% after background subtraction), the 59 sequences are not. This fact could imply that the 39 enhancer acts first and its stimulatory activity is required for the subsequent action of the 59 enhancing sequences. It is important to note that the transfer of both sequence elements into the sequence context of REI- nonpermissive uORF4 converts it into a REI-permissive uORF [19]. Hence the mechanism of their combined action appears to be general, not specific to uORF1 only. The 59 sequences of uORF1 contain at least three REI-promoting elements one of which operates in an a/TIF32-NTD–dependent manner Whereas the molecular mechanism by which the 39 enhancer promotes REI is unknown, our recent genetic epistasis analysis suggested that the 59 sequences (in segment A) emerging from the 40S mRNA exit channel promote REI by interacting with the NTD of a/TIF32 upon termination on the uORF1 stop codon. This interaction was proposed to stabilize association of the post- termination 40S subunit with the GCN4 mRNA so that it could resume scanning for REI downstream [10]. Partial deletions of the 59 sequences in the GCN4-lacZ construct containing solitary uORF1 had severe deleterious effects on efficiency of REI in the wt a/TIF32 background but not in the a/tif32D cells expressing a viable a/tif32-D8 allele lacking sequences encoding the extreme N- terminal 200 amino acid residues. Given that the 59 enhancing sequences comprise a rather long stretch of ,160 nt, however, it is fairly unlikely that such a long segment contacts eIF3 bound to the 40S as a whole. In fact, previously published data suggested that it may consist of at least two critical elements, as deletions of 40, 80 and 120 nt from nt 221 upstream reduced the GCN4-lacZ expression by a similar fold (from 2.5- to 3-fold), whereas the largest deletion of 160 nt resulted in ,6-fold reduction [14]. In order to precisely map the minimal region(s) responsible for the REI-promoting role of the uORF1’s 59 sequences that work in concert with the a/TIF32-NTD, the 59 sequences were progres- sively deleted (beginning at a position 216 nt relative to the uORF1 AUG codon) in a GCN4-lacZ construct containing solitary uORF1 (Figure 2A). For example, constructs DEL6 and DEL36 had internal deletions of 6 nt (from 216 to 221) and 36 nt (from 216 to 251), respectively. As a specific background control, the 4- 4-1-1 construct devoid of the entire 59 enhancing sequences (defined in Figure 1C) was routinely used (bg*). All deletion constructs were expressed in both the a/TIF32 wt and a/tif32-D8 PLoS Genetics | www.plosgenetics.org 4 July 2011 | Volume 7 | Issue 7 | e1002137