Abstract
Conserved ribosomal proteins frequently harbor additional segments in eukaryotes not found in bacteria, which could facilitate eukaryotic-specific reactions in the initiation phase of protein synthesis. Here we provide evidence showing that truncation of the N-terminal domain (NTD) of yeast Rps5 (absent in bacterial ortholog S7) impairs translation initiation, cell growth and induction of GCN4 mRNA translation in a manner suggesting incomplete assembly of 48S preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5. Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site. We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNAiMet to promote a functional 48S PIC.
Highlights
Eukaryotic translation initiation is a complex process involving multiple steps [1]
Regulation of GCN4 translation is exerted via a reinitiation process involving four small upstream open reading frames preceding the GCN4 ORF and is known to be a sensitive indicator of translation initiation defects in vivo (for a review, see [26])
We further considered a second possibility to account for the strong Gcn− phenotype of the rps5-Δ1-46 strain, wherein the mutant rps5-Δ1-46 40S subunits would exhibit a reduced rate of scanning that allows the ternary complex (TC) to be acquired before the reinitiating ribosomal subunits bypass uORF4, thereby increasing reinitiation at uORF4 and producing an equivalent reduction in reinitiation at GCN4
Summary
Eukaryotic translation initiation is a complex process involving multiple steps [1]. For the majority of cellular mRNAs it starts with the recruitment of Met-tRNAiMet to the 40S ribosomal subunit by eukaryotic-specific initiation factor eIF2 [1]. In cooperation with initiation factors eIF3, 1/1A, the eIF2·GTP·Met-tRNAiMet ternary complex (TC), binds the 40S ribosomal subunit yielding the 43S preinitiation complex (PIC) [1]. The 43S complex binds to the 5 -end of mRNA and scans in search of the initiation codon to form the 48S PIC. Following recognition of the start codon and eIF5-induced irreversible hydrolysis of eIF2-bound Guanosine-5’-triphosphate (GTP), eIF5B promotes joining of the 40S and 60S subunits and the elongation process begins [1]. While recent studies have yielded detailed insights into the mechanism of translation initiation, many details of the process remain unknown. The exact placement and orientation of initiation factors on the ribosomal surface, structural rearrangements accompanying various steps of initiation, the role played by ribosomal proteins, the timing (and kinetics) of factor association and release and, the exact architecture of the 43S and 48S PICs are either unknown or just beginning to emerge [2,3]
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