Limits...
eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

Pisareva VP, Pisarev AV - Nucleic Acids Res. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA andrey.pisarev@downstate.edu.

Show MeSH

Related in: MedlinePlus

eIF5 and eIF5B stimulate 48S IC formation and support correct placement of mRNA during ribosomal scanning. (A) Structures of TC, AC and AG mRNAs. (B–E) Toeprint analysis of 48S IC assembly on (B) TC, AC and AG mRNAs, (C, D) TC mRNA and (E) native β-globin mRNA. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences. Toeprint assays are supplemented with quantification which shows yield of 48S IC on each initiation codon calculated as percentage of toeprint signal to summarized signal in lane (n ≥ 3, SD < 14%).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231746&req=5

Figure 2: eIF5 and eIF5B stimulate 48S IC formation and support correct placement of mRNA during ribosomal scanning. (A) Structures of TC, AC and AG mRNAs. (B–E) Toeprint analysis of 48S IC assembly on (B) TC, AC and AG mRNAs, (C, D) TC mRNA and (E) native β-globin mRNA. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences. Toeprint assays are supplemented with quantification which shows yield of 48S IC on each initiation codon calculated as percentage of toeprint signal to summarized signal in lane (n ≥ 3, SD < 14%).

Mentions: To get insights into the mechanism of stimulation, we tested the effect of eIF5 and eIF5B in the 48S IC formation on other mRNAs whose initiation potential deviates from optimal. One subset of such mRNAs contains the initiation codon in the non-optimal initiation context. It has been shown that the scanning 43S PIC may pass the initiation codon in the non-optimal nucleotide context without the formation of the 48S IC (32). Purines in the ‘−3’ and ‘+4’ positions relative to the AUG codon are the key nucleotides in the context. We decided to test native eIF5 and eIF5B in the reconstituted system on model mRNAs comprising the single-stranded 5′-UTR followed by two initiation codons separated by the 12-nt single-stranded region (Figure 2A). The second start codon is linked with the β-glucuronidase ORF. All three mRNAs contain the second AUG codon in the optimal context, whereas the first AUG codon is in the non-optimal (TC mRNA), suboptimal (AC mRNA) or optimal (AG mRNA) context depending on the presence of two pyrimidines, one purine and one pyrimidine, or two purines in the ‘−3’ and ‘+4’ positions, respectively (32). 48S IC formed predominantly on the second AUG and weakly on the first AUG of TC mRNA (Figure 2B, lanes 1 and 2), distributed equally between both AUG codons of AC mRNA (Figure 2B, lanes 4 and 5) or assembled almost exclusively on the first AUG of AG mRNA (Figure 2B, lanes 7 and 8). Native eIF5 and eIF5B together resulted in the higher yield of 48S IC on the first codon of all three mRNAs (Figure 2B, lanes 3, 6 and 9). The highest stimulation was noted in the case of non-optimal context of the first codon presented in the TC mRNA. Therefore, we chose the TC mRNA over other mRNAs for the subsequent experiments.


eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

Pisareva VP, Pisarev AV - Nucleic Acids Res. (2014)

eIF5 and eIF5B stimulate 48S IC formation and support correct placement of mRNA during ribosomal scanning. (A) Structures of TC, AC and AG mRNAs. (B–E) Toeprint analysis of 48S IC assembly on (B) TC, AC and AG mRNAs, (C, D) TC mRNA and (E) native β-globin mRNA. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences. Toeprint assays are supplemented with quantification which shows yield of 48S IC on each initiation codon calculated as percentage of toeprint signal to summarized signal in lane (n ≥ 3, SD < 14%).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231746&req=5

Figure 2: eIF5 and eIF5B stimulate 48S IC formation and support correct placement of mRNA during ribosomal scanning. (A) Structures of TC, AC and AG mRNAs. (B–E) Toeprint analysis of 48S IC assembly on (B) TC, AC and AG mRNAs, (C, D) TC mRNA and (E) native β-globin mRNA. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences. Toeprint assays are supplemented with quantification which shows yield of 48S IC on each initiation codon calculated as percentage of toeprint signal to summarized signal in lane (n ≥ 3, SD < 14%).
Mentions: To get insights into the mechanism of stimulation, we tested the effect of eIF5 and eIF5B in the 48S IC formation on other mRNAs whose initiation potential deviates from optimal. One subset of such mRNAs contains the initiation codon in the non-optimal initiation context. It has been shown that the scanning 43S PIC may pass the initiation codon in the non-optimal nucleotide context without the formation of the 48S IC (32). Purines in the ‘−3’ and ‘+4’ positions relative to the AUG codon are the key nucleotides in the context. We decided to test native eIF5 and eIF5B in the reconstituted system on model mRNAs comprising the single-stranded 5′-UTR followed by two initiation codons separated by the 12-nt single-stranded region (Figure 2A). The second start codon is linked with the β-glucuronidase ORF. All three mRNAs contain the second AUG codon in the optimal context, whereas the first AUG codon is in the non-optimal (TC mRNA), suboptimal (AC mRNA) or optimal (AG mRNA) context depending on the presence of two pyrimidines, one purine and one pyrimidine, or two purines in the ‘−3’ and ‘+4’ positions, respectively (32). 48S IC formed predominantly on the second AUG and weakly on the first AUG of TC mRNA (Figure 2B, lanes 1 and 2), distributed equally between both AUG codons of AC mRNA (Figure 2B, lanes 4 and 5) or assembled almost exclusively on the first AUG of AG mRNA (Figure 2B, lanes 7 and 8). Native eIF5 and eIF5B together resulted in the higher yield of 48S IC on the first codon of all three mRNAs (Figure 2B, lanes 3, 6 and 9). The highest stimulation was noted in the case of non-optimal context of the first codon presented in the TC mRNA. Therefore, we chose the TC mRNA over other mRNAs for the subsequent experiments.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA andrey.pisarev@downstate.edu.

Show MeSH
Related in: MedlinePlus