Kinetic and thermodynamic analysis of the role of start codon/anticodon base pairing during eukaryotic translation initiation

Abstract

Start codon recognition is a crucial event in the initiation of protein synthesis. To gain insight into the mechanism of start codon recognition in eukaryotes, we used a yeast reconstituted initiation system to isolate the step of Met-tRNA i d eIF2 d GTP ternary complex (TC) binding to the 40S subunit. We examined the kinetics and thermodynamics of this step in the presence of base changes in the mRNA start codon and initiator methionyl tRNA anticodon, in order to investigate the effects of base pairing and sequence on the stability of the resulting 43S d mRNA complex. We observed that the formation of three base pairs, rather than their identities, was the key determinant of stability of TC binding, indicating that nothing is inherently special about the sequence AUG for this step. Surprisingly, the rate constant for TC binding to the 40S subunit was strongly codon dependent, whereas the rate constant for TC dissociation from the 43SdmRNA complex was not. The data suggest a model in which, after the initial diffusion-limited encounter of TC with the 40S subunit, the formation of three matching start codon/anticodon base pairs triggers a conformational change that locks the complex into a stable state. This induced-fit mechanism supports the proposal that initiation codon recognition by the 43S complex induces a conformational change from an open state to a closed one that arrests movement along the mRNA.

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

“…Notably, the AUG uORF of YGR210C had the largest impact on the reporter, whereas NCC uORFs had smaller effects. This is consistent with expectations, given the weaker recruitment of ribosomes to NCC start codons (Kolitz et al 2009). The AUG and NCC uORFs we identified appear to function as both positive and negative regulators.…”

“…Indeed, AUG uORFs were more occupied than NCC uORFs (Fig. 4D), consistent with their known higher rates of translation initiation (Kolitz et al 2009). Finally, we found that both AUG and NCC uORFs had higher translation efficiency under two stress conditions (rapamycin and diamide treatment) ( In S. cerevisiae, nearly 45% of uORFs initiate with AUG, compared with ∼15% in S. paradoxus and S. uvarum.…”

“…Intriguingly, there have been hints that uORFs may be more common than initially believed. Translation can initiate (mostlikely using tRNA i Met ) at near cognate codons (NCCs)-sequences that differ from the canonical AUG start codon by one nucleotide (Zitomer et al 1984;Peabody 1989;Ivanov et al 2008Ivanov et al , 2011Kolitz et al 2009;Starck et al 2012;Kochetov et al 2013;Gao et al 2014). Thus, many NCC uORFs may be lying hidden in eukaryotic genomes.…”

“…3C). For each uORF, we compared levels of protein (YFP/ mCherry) and RNA (qPCR) from WT and nonfunctional "AGG" start codon (Kolitz et al 2009) mutant plasmids (ΔTIS) (Fig. 3C).…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…As the start codon mutations in the mRNA mainly affected the on rate for Met-tRNA i Met binding, and not the off rate, it was proposed that 48S PIC formation is accompanied by a conformational change that locks in Met-tRNA i Met binding. Accordingly, in this closed state, Met-tRNA i Met is stably bound to the 40S subunit and fixed on the translation start codon of the mRNA (Kolitz et al 2009). …”