Constraints on reinitiation of translation in mammals

Abstract

The efficiency of reinitiation in mammalian translation systems depends in part on the size and arrangement of upstream open reading frames (upORFs). The gradual decrease in reinitiation as an upORF is lengthened, confirmed here using a variety of sequences, might reflect time-dependent loss of protein factors required for reinitiation. Consistent with the idea that the duration of elongation is what matters, reinitiation was nearly abolished when a pseudoknot that causes a pause in elongation was inserted into a short upORF. Control experiments showed that this transient pause in elongation had little effect on the final protein yield when the pseudoknot was moved from the upORF into the main ORF. Thus, the deleterious effect of slowing elongation is limited to the reinitiation mode. Another aspect of reinitiation investigated here is whether post-termination ribosomes can scan backwards to initiate at AUG codons positioned upstream from the terminator codon. Earlier studies that raised this possibility may have been complicated by the occurrence of leaky scanning along with reinitiation. Re-examination of the question, using constructs that preclude leaky scanning, shows barely detectable reinitiation from an AUG codon positioned 4 nt upstream from the terminator codon and no detectable reinitiation from an AUG codon positioned farther upstream. These experiments carried out with synthetic transcripts help to define the circumstances under which reinitiation may be expected to occur in the growing number of natural mRNAs that deviate from the simple first AUG rule.

Figure 1

The size of the upstream minicistron affects the efficiency of reinitiation. A 13-codon upORF designated 13X was inserted into the basic CAT mRNA sequence (topmost line) at the point shown. Constructs in which the minicistron was lengthened to 23 or 33 codons were obtained by reiterating a 30 nt segment (underlined in red) of upORF 13X. Lanes 2–4 in the polyacrylamide gel show the yield of CAT protein from this set of mRNAs. In similar fashion, an alternative upORF, designated 13Y, was expanded by reiterating the 30 nt segment underlined in blue, producing the mRNAs tested in lanes 5–7. For comparison, the yield of CAT from an mRNA that has no upORF is shown in lane 1.

Figure 2

The sequence used to expand the upstream minicistron does not impair elongation per se. Translation of mRNAs from series X (described in Fig. 1) in which the upORF is 13, 23 or 33 codons long is shown in lanes 1–3, respectively. In the accompanying control mRNAs, 13 (lane 4), 23 (lane 5) or 33 codon (lane 6) upORF has been fused with the CAT coding domain. This was accomplished by changing the terminator codon of the upORF from UAA to UAC and inserting one extra base to adjust the reading frame. The resulting N-terminally extended forms of CAT (labeled preCAT) are distinguishable by PAGE under the conditions described in Materials and Methods.

Figure 3

Sequences of pseudoknot-containing mRNAs used in this study. Downstream from the BamH1 site (line 1) these mRNAs are identical to the transcript shown in full in Figure 1. In line A, the sequences highlighted in blue and red form a pseudoknot close to the 5′ end of the mRNA and far upstream from the CAT coding domain (construct 2 in Fig. 4A). The mRNAs depicted in lines B and C contain a 17-codon upORF defined by the underlined AUG and UAG codons. Within this upORF, a pseudoknot forms in the mRNA depicted in line C (construct 7 in Fig. 4A). The potential for base pairing within the upORF has been minimized in line B (construct 8 in Fig. 4A), which thus serves as a control for construct 7. Line D depicts another control in which the pseudoknot occurs, not within an upORF, but within the main coding domain (construct 5 in Fig. 4A). This was achieved by mutating the terminator codon of the upORF. As a result, ribosomes that initiate translation at the upstream AUG codon continue uninterrupted through the CAT coding domain, producing an N-terminally extended ‘preCAT’ protein. Because the pseudoknot in lines C and D begins 13-nt downstream from the AUG codon, assembly of an initiation complex is not impeded; the pseudoknot thus positioned is a barrier to only the elongation phase of translation. Deletion of 6 nt (underlined in line D) moves the pseudoknot closer to the start codon and thus blocks the initiation (scanning) phase of translation (construct 4 in Fig. 4A).

Figure 4

Reinitiation is inhibited by a pseudoknot in the upORF that slows elongating ribosomes. The pseudoknot depicted in the boxed insert was positioned in the 5′ untranslated region (constructs 2 and 3), in the main coding domain (constructs 4 and 5) or in an upORF (construct 7). The polyacrylamide gels in (B) and (C) show protein yields from these structure-containing mRNAs and from unstructured control transcripts (constructs 1 and 8). The numeral preceding each mRNA in (A) matches the number of the lane in which that mRNA was tested (B and C). Construct 6 is the same as construct 5. Coding domains, which are not drawn to scale, are shown as filled boxes. The actual sequences of the mRNAs are given in Figure 3. Capped mRNAs were translated as usual at 25°C in reticulocyte lysate supplemented with [³H]leucine and 2 mM Mg²⁺.

Figure 5

Test of whether ribosomes can backup to reinitiate. In addition to a nine-codon upORF (underlined in red), each mRNA has an AUG codon (AUG^preCAT highlighted in black) upstream from and in the same reading frame as CAT. Ribosomes that engage the mRNA depicted in the first line would be expected to translate the upORF and then move forward 11 nt to reinitiate translation at AUG^preCAT. This mRNA was tested in lane 2 of the autoradiogram. With the mRNAs depicted in the second and third lines, ribosomes that translate the upORF would have to backup 7 or 13 nt to reach AUG^preCAT. Translation of these mRNAs was tested in lanes 5 and 8. The relative yields of CAT and preCAT proteins indicate the extent to which ribosomes move forward or backward after translating the upORF. For the positive controls in lanes 3, 6 and 9, the start codon of the upORF was mutated, thus making AUG^preCAT the first AUG in the mRNA. In the negative controls (lanes 4, 7 and 10), the indicated mutation of the terminator codon from UAA to UCA causes the upORF (in reading frame –2) to overlap the CAT coding domain for a distance of 133 nt. With reinitiation thus precluded in the negative controls, their failure to produce CAT or preCAT proteins establishes that there is no leaky scanning: all ribosomes recognize the upORF start codon. Thus, reinitiation is the sole mechanism for producing preCAT and/or CAT proteins in lanes 2, 5 and 8. Electrophoresis conditions were adjusted to ensure that the small ‘out-of-frame’ polypeptides (lanes 4, 7 and 10) were retained on the gel. This might be why the various forms of preCAT, differing in length by 7–9 amino acids, are not resolved from one another.