Why is start codon selection so precise in eukaryotes?

Abstract

Translation generally initiates with the AUG codon. While initiation at GUG and UUG is permitted in prokaryotes (Archaea and Bacteria), cases of CUG initiation were recently reported in human cells. The varying stringency in translation initiation between eukaryotic and prokaryotic domains largely stems from a fundamental problem for the ribosome in recognizing a codon at the peptidyl-tRNA binding site. Initiation factors specific to each domain of life evolved to confer stringent initiation by the ribosome. The mechanistic basis for high accuracy in eukaryotic initiation is described based on recent findings concerning the role of the multifactor complex (MFC) in this process. Also discussed are whether non-AUG initiation plays any role in translational control and whether start codon accuracy is regulated in eukaryotes.

Keywords: archaeal translation; bacterial translation; eukaryotic translation; initiation factors; multifactor complex; non-AUG initiation; ribosome; start codon fidelity; start codon selection; translation initiation.

Figure 1. Control of GUG-initiated translation through dynamic mRNA conformational changes. (A) The replication region of ColIb-P9 plasmid. The horizontal line represents the DNA of the ColIb-P9 replication region. Boxes denote coding regions for Inc, RLP and Rep and the origin of replication (ori). The short leftward arrow below denotes Inc RNA (antisense), while the long rightward arrow denotes Rep mRNA, starting from its promoter, Prep. Green circles on the transcripts denote the GGCG (filled) and CGCC (open) sequences, which interact together during rep gene regulation. The base-pairing between these sequences in Rep mRNA forms a pseudoknot with the Inc target stem-loop, which then allows rep translation (+ sign). The CGCC sequence (green open circle) on Inc RNA binds the GGCG sequence (green filled circle) on Rep mRNA, inhibiting pseudoknot formation (- sign). Inc RNA also represses RLP translation (- sign). (B) Inhibitory secondary structure found in the translation initiation region of the rep genes in ColIb-P9. Oragne boxes, GUG start codon and SD sequence (also with asteriska). Underline, RLP stop codon. Green box, the CGCC sequence forming a pseudoknot. (C) Models for the control of ColIb-P9 rep translation by competition between pseudoknot formation and antisense RNA binding. Panels a-d denote a conformation state of Rep mRNA, with green boxes denoting the GGCG (filled box) and CGCC (open box) sequences with the potential to form a pseudoknot. Bracket indicates the Rep mRNA region complementary to Inc RNA. Orange boxes are the SD and the GUG start codon for rep. Thin arrows on the mRNA indicate the regions that get translated in each state. Black filled and open circles denote the start and stop codons of RLP.

Figure 2. Eukaryotic 48S PIC. Schematics on the bottom depict the eukaryotic 48S PIC formed at the 5` end of mRNA (horizontal line) with the AUG codon (box) ahead. The m⁷G cap, shown by an orange small circle, is bound by eIF4F in teal. The translation initiation multifactor complex (MFC) bridges eIF4F and the 40S SSU, the large pale green circle with while letters indicating E, P, and A-sites. Met-tRNA_i^Met, shown as a plug, is attached through the eIF2 component of the MFC, but not firmly linked to the P-site during scanning (Pout). Arrow indicates the direction of scanning. The 43S PIC is made of the 40S SSU, MFC, eIF1A, and Met-tRNA_i^Met. On top, eIF1 (pyramid) bound near the P-site blocks Met-tRNA_i^Met (plug) binding to the P-site together with other MFC partners, until it base-pairs to the AUG codon. The thick stopped bar indicates the physical block of the P-site formed by the presented part of the MFC. Dotted lines denote the interactions of eIF1 with other MFC partners shown by circles.

Figure 3. Translation factor control of the PIC conformational change, coupled to AUG recognition. (A) The open (left) and closed (right) states of the 48S PIC are depicted with the 40S SSU (large circle with E, P and A-sites, different colors indicating distinct states) bound to initiation factors (small circles with numbers corresponding to each eIF). In the open state (left), the decoding site is “open,” allowing mRNA (curved thick line with 5′ and 3′ ends indicated) to slide along the mRNA-binding channel. Met-tRNA_i^Met (plug) is tilted to imply that it is out of the P-site. eIF1 (circle numbered 1) prevents Met-tRNA_i^Met paired with the non-AUG codon from binding the P-site. In the closed state (right), eIF1 is released (thick horizontal arrow), allowing Met-tRNA_i^Met paired with the AUG codon to bind the P-site. The Met-tRNA_i^Met is positioned vertically to imply the Pin state (near the P/I configuration^18,20). (B) The conformational change is explained by the competition between eIF1 (circle) and the Met-tRNA_i^Met (plug):base triplet pair (UUG or AUG) for the P-site (labeled white on a part of green circles representing the 40S SSU). (C) MFC partners and eIF1A tails serve to stabilize each state, tightly coupling AUG recognition and commitment to initiation. In the open state (left), indicated MFC partners (circles) and eIF1A-NTT (thick red line) assist eIF1 to block tRNA_i^Met paired to UUG (representing a non-AUG codon) from binding the P-site. eIF1A-CTT (thick red line) destabilizes tRNA_i^Met binding to the P-site, contributing to the maintenance of the open state. In right, eIF2β-NTT (thick maroon line) plays the major role in the shift to the closed state by binding eIF5-CTD and disrupting the eIF1 linking to the PIC. These interactions promote eIF1 release. eIF1A-NTT (thick red line) stabilizes tRNA_i^Met binding to the P-site by direct interaction with tRNA_i^Met and the 40S P-site.

Figure 4. Possible mechanisms of translational control by regulated initiation stringency. Typical coding structures of mRNA (horizontal lines) are depicted with the main ORF starting with the canonical AUG codon (longer box to the right). Black and red arrows indicate AUG- and non-AUG-initiated translation, respectively. mRNA translation patterns in the normal stringent mode (A) and the hypothetical non-stringent mode (B) are depicted. In (A), dotted boxes depict non-AUG-initiated uORFs that are not translated due to the normal, stringent mode of initiation. The main ORF is translated predominantly. In (B), panels 1–4 describe four patterns of regulation achieved by non-AUG-initiation. In panel 1, non-AUG-initiated uORF is permissive to downstream re-initiation, allowing the main ORF translation. In panel 2, non-AUG-initiated uORF is non-permissive to downstream re-initiation. The translation of the main ORF is only possible by the ribosome that had leaky-scanned the uORF (dotted line). In panel 3, the upstream permissive uORF allows downstream re-initiation of the second non-permissive uORF “or” the main ORF. When eIF2 TC abundance and recruitment are normal, the second uORF is re-initiated, inhibiting the main ORF translation. When eIF2 TC is limited or its ribosome binding is inhibited, the re-initiation is delayed and occurs at the main ORF. In panel 4, thick lines below mRNA schematics denote proteins encoding by the mRNA, with boxes indicating N-terminal functional segments added by the in-frame non-AUG initiation. Though not depicted here, there are myriads of mRNAs with AUG-initiated uORFs that are translated in the stringent mode, playing important roles in translational control. See text for details.