The Genomic Landscape of Compensatory Evolution Laboratory selection experiment explains how organisms compensate for the loss of genes during evolution, and reveals the deleterious side-effects of this process when adapting to novel environments.

Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5 of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5 enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.[Keywords: Translation initiation; reinitiation; eIF3; 40S ribosomal subunit; GCN4; short uORF] Supplemental material is available at http://www.genesdev.org.

Despite the recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor 3 (eIF3), there is still only little known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic domain (NTA) is held in the helix α1 – loop L5 hydrophobic pocket of the human eIF3b-RRM. Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S-occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half (NTD) of eIF3j/HCR1 containing the NTA motif is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its NTD only, or mutating the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed pre-initiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning pre-initiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together we propose that eIF3j/HCR1 closely co-operates with eIF3b/PRT1-RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1–3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein–protein binding domain in mediating protein–RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.

Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.