Abstract
48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5′-end of messenger RNA (mRNA) and scans its 5′-untranslated region (5′-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons—following structured 5′-UTRs, in bad AUG context, within few nucleotides from 5′-end of mRNA and CUG start codon—is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.
Highlights
Eukaryotic translation can be divided into initiation, elongation, termination and ribosomal recycling steps
multifactor complex (MFC) together with eIF1A, which is the ortholog of bacterial IF1, bind 40S ribosomal subunit with the formation of 43S pre-initiation complex (43S PIC). eIF3 mediates the attachment of 43S PIC to the 5 -end of messenger RNA (mRNA). eIF1 and eIF1A synergistically bind to the 40S subunit [5]. eIF1 occupies the P site and the folded body of eIF1A binds in the A site of the 40S subunit, but its unstructured, long N- and C-terminal tails reach into the P site [6,7]. eIF1 and eIF1A together upon binding promote the rearrangement of the 40S subunit from a closed scanning-arrested to an open scanning-competent state [8]
The reconstituted system comprising eukaryotic initiation factor (eIF) 1, 1A, 2, 3, 40S ribosomal subunits, Met-tRNAiMet and DHX29 promotes a 48S initiation complex (48S IC) formation on the model Stem1 mRNA consisting of single-stranded 5 -untranslated region (5 -UTR) with centrally introduced stem ( G = −5.5 kcal/mol) linked with -glucuronidase open reading frame (ORF) (Figure 1A)
Summary
Eukaryotic translation can be divided into initiation, elongation, termination and ribosomal recycling steps. TC associates with eIF1, eIF3 and eIF5 in the multifactor complex (MFC). EIF1 and eIF1A together upon binding promote the rearrangement of the 40S subunit from a closed scanning-arrested to an open scanning-competent state [8]. MFC together with eIF1A, which is the ortholog of bacterial IF1, bind 40S ribosomal subunit with the formation of 43S pre-initiation complex (43S PIC). Such a conformational change underlies the mechanism by which these factors secure the fidelity of initiation codon selection. GDP and Pi remain associated with eIF2 until the initiation codon recognition [9]
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