Mechanisms and regulation of translation initiation

b. Scanning and AUG selection

Our analyses of the prt1-1 mutation in b/PRT1 and of mutations in eIF1A have implicated specific residues in these factors that appear to promote scanning during reinitiation on GCN4 mRNA. The prt1-1 mutation in eIF3b leads to accumulation of bulk 48S PICs, signifying a block in the pathway between binding of 43S PICs to mRNA and 60S subunit joining. Assaying different GCN4-lacZ reporters indicates that prt1-1 eliminates the ability of 40S ribosomes scanning downstream from uORF1 to bypass uORF4 and reinitiate at GCN4 when TC levels are reduced by eIF2a phosphorylation—producing a strong Gcn^- phenotype. The simplest explanation for these results is that prt1-1 reduces the rate of scanning, increasing the time required to traverse the uORF1-uORF4 interval and thereby compensates for the delay in TC loading produced by eIF2(aP) ²⁰ (Fig. 7A).

In a related development, we identified Ala substitution mutations in the helical domain of eIF1A (residues 98-101) that confer reduced eIF2 association with native PICs and slow-growth (Slg^-), which both are rescued by hc-TC. Despite the impaired 40S binding of TC, the 98-101 mutation produces a Gcn^- phenotype rather than the expected Gcd^- phenotype. We suggested that 98-101 also confers slower scanning between uORFs 1 and 4, which compensates for the delay in TC loading and restores efficient reinitiation at uORF4, with impaired induction of GCN4 (Gcn^-). In collaboration Tatyana Pestova’s lab, we obtained evidence supporting this model by determining the effect of 98-101 on scanning in a reconstituted mammalian system in which inhibition of primer extension (toe-printing) is used to map the locations of scanning PICs on specific mRNAs. We found that yeast eIF1A substitutes for the mammalian eIF1A in this assay and that 98-101 reduces the ability of PICs to both migrate from the cap and to bypass an upstream GUG (Fig. 8).

We proposed that aberrant GUG recognition results from a higher dwell time for each triplet in the P-site that increases the chance for non-AUG selection before scanning resumes. In agreement with this, 98-101 increases initiation of a lacZ reporter with a UUG start codon (Sui- phenotype) and decreases leaky scanning of the AUG at GCN4 uORF1 in vivo—both consistent with a higher probability of start codon selection versus continued scanning. Strikingly, the DC truncation and 131,133 point mutations in the eIF1A-CTT likewise increase initiation at UUG and AUGs in vivo, and DC confers defective scanning and elevated GUG selection in the mammalian in vitro system. We concluded that the CTT and helical domain of eIF1A promote  scanning, and that a slower rate of scanning in the eIF1A mutants is responsible for both increased UUG initiation and impaired derepression of GCN4 translation ^10,11.

By measuring the kinetics of eIF1A dissociation from reconstituted 48S PICs, Lorsch and colleagues found that eIF1A binding to the PIC is strengthened (resulting in slower dissociation and less rotational freedom of its CTT), when AUG occupies the P-site and eIF5 is present in the complexes. As both the DC truncation of eIF1A and the SUI5 mutation in eIF5 strengthen eIF1A binding at UUG in this assay, and both mutations increase UUG initiation in vivo (Sui^- phenotypes), they proposed that tighter binding of eIF1A characterizes the closed, scanning arrested conformation of the initiation complex at the start codon ⁴ (Fig. 6a).

In collaboration with Lorsch’s group, we showed that the Sui^- eIF1A-CTT mutation 131,133 behaved similarly to DC in strengthening eIF1A binding at UUG, whereas mutations in the unstructured NTT of eIF1A (residues 7-11 or 17-21) have the opposite effect and weaken eIF1A binding with UUG or AUG in the P-site (Fig. 9).

Remarkably, these NTT mutations suppress the Sui^- phenotypes of mutations in eIF5 (SUI5)and eIF2b (SUI3-2) and increase leaky scanning of the GCN4 uORF1 AUG (Fig. 10)—both consistent with a reduced probability of start codon selection vs. continued scanning.

Thus, the eIF1A CTT and NTT mutations have opposing effects on start codon selection and binding of eIF1A to the PIC, indicating that the strength of eIF1A association with the PIC is an important determinant of start codon selection in vivo ¹¹.

Several lines of evidence converged recently indicating that eIF1 is a negative regulator of initiation at non-AUG codons. We found that overexpression of eIF1 suppresses the increased UUG initiation in various Sui^- mutants ⁵. Pestova’s group showed that eIF1 blocks PIC assembly at non-AUG triplets in the reconstituted mammalian system ⁶ and eIF1 also restrains eIF5-stimulated GTP hydrolysis at non-AUGs ²¹. They mapped the location of eIF1 near the P-site and suggested that it promotes an open conformation of the PIC that is conducive to scanning and restricts base-pairing of Met-tRNA_i^Met with non-AUG triplets ²². Using the reconstituted yeast system, Lorsch’s group showed that AUG in the P-site evokes a rapid conformational change that increases separation between eIF1 and the eIF1A CTT (detected as loss of FRET between fluorescently tagged forms of eIF1A and eIF1) followed by dissociation of eIF1 from the 40S subunit ²³. They also showed that P_i release is stimulated much more than hydrolysis of GTP to GDP-P_i in TC by an AUG in the P-site, that the kinetics of eIF1 dissociation and P_i release are similar, and that the G107R mutation in eIF1 similarly reduces the rates of both reactions ⁷. This all suggested a model wherein eIF1 blocks non-AUG selection by preventing P_i release, in addition to its other functions in restraining eIF5 GAP function and promoting scanning. All three eIF1 functions would be eliminated when AUG base-pairs with Met-tRNA_i^Met, as this triggers eIF1 dissociation from the PIC ²³.

In collaboration with Lorsch’s and Pestova’s groups, we obtained strong support for the idea that dissociation of eIF1 from the 40S subunit is a key step in start codon selection in vivo. We showed that Sui^- eIF1 mutations D83G, Q84P, and 93-97 all decrease eIF1 affinity for 40S subunits and both the Sui^- phenotypes and impaired 40S binding of eIF1 are partially corrected by overexpressing the mutant proteins in vivo. Importantly, the 93-97 mutation elevates the rates of both eIF1 dissociation and Pi release from eIF2-GDP-P_i in reconstituted PICs (Fig. 11).

All 3 eIF1 mutations also increase selection of non-AUGs in the reconstituted mammalian system independent of GTP hydrolysis. Remarkably, the eIF1A NTT mutation FL-17-21 that suppresses UUG initiation in Sui^- mutants (Ssu^- phenotype) decreases (rather than increases) the rate of eIF1 release from reconstituted initiation complexes (Fig. 11). These results indicate that release of eIF1 from the 40S subunit is a critical step in AUG selection in vivo, allowing P_i release and the transition to a closed, scanning-arrested conformation of the PIC, that is modulated by eIF1A ⁹. We have also identified Sui^- and Ssu^- mutations in the eIF3c/NIP1 NTT (Fig. 3), that weaken interactions of this eIF3 domain with eIF1 and eIF5, suggesting that eIF3 helps to coordinate the opposing functions of eIF1 and eIF5 in AUG selection ⁵.

Finally, in collaboration with Mercedes Tamame, we characterized a Gcd^- mutation in residue G76 of the 60S subunit protein RPL33A that impairs 40S-60S subunit joining in vivo. The G76R mutation leads to leaky scanning of the inhibitory uORF4 in GCN4 mRNA, presumably by reassembled PICs containing the TC, to elicit derepression of GCN4 translation (Gcd^- phenotype). Interestingly, overexpressing tRNA_i^Met suppresses the Gcd^- phenotype of rpl33a-G76R, suggesting that Met-tRNA_i^Met dissociates from 48S PICs stalled by the delay in subunit joining at the uORF4 start codon, and that this abortive event can be reversed by mass-action to prevent leaky scanning of the uORF ²⁴.