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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.

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eIF5 and eIF5B are essential for stimulation of 48S IC formation. (A) Structure of Stem1 mRNA. (B, D and E) Toeprint analysis of 48S IC assembly on Stem1 mRNA in the presence of (B) RRL fraction or (D and E) different combinations of eIF5 and eIF5B. Initiation codon and position of assembled 48S IC are indicated. Lanes C/T/A/G depict corresponding DNA sequence. Toeprint assays are supplemented with quantification which shows yield of 48S IC calculated as percentage of toeprint signal to summarized signal in lane. Number of replicated experiments for quantification is at least three (n ≥ 3) and standard deviation is less than 14% (SD < 14%). (C) Left panel: purification scheme for eIF5 and eIF5B; right panel: purified eIF5 and eIF5B resolved by SDS-PAGE.
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Figure 1: eIF5 and eIF5B are essential for stimulation of 48S IC formation. (A) Structure of Stem1 mRNA. (B, D and E) Toeprint analysis of 48S IC assembly on Stem1 mRNA in the presence of (B) RRL fraction or (D and E) different combinations of eIF5 and eIF5B. Initiation codon and position of assembled 48S IC are indicated. Lanes C/T/A/G depict corresponding DNA sequence. Toeprint assays are supplemented with quantification which shows yield of 48S IC calculated as percentage of toeprint signal to summarized signal in lane. Number of replicated experiments for quantification is at least three (n ≥ 3) and standard deviation is less than 14% (SD < 14%). (C) Left panel: purification scheme for eIF5 and eIF5B; right panel: purified eIF5 and eIF5B resolved by SDS-PAGE.

Mentions: The reconstituted system comprising eIFs 1, 1A, 2, 3, 40S ribosomal subunits, Met-tRNAiMet and DHX29 promotes a 48S IC formation on the model Stem1 mRNA consisting of single-stranded 5′-UTR with centrally introduced stem (ΔG = −5.5 kcal/mol) linked with β-glucuronidase open reading frame (ORF) (Figure 1A). This mRNA has been already described and imitates the one with the moderately structured 5′-UTR (19). Despite the presence of DHX29 helicase, the efficiency of 48S IC assembly in the reconstituted system on mRNAs with structured 5′-UTR is still much lower than their translation level in the RRL (19; data not shown). Therefore, to identify the activities improving the 48S yield on Stem1 mRNA, we fractionated RRL and tested fractions in the system. After several fractionation steps, we had a phosphocellulose (P-11) fraction with a dozen of proteins which stimulated 48S IC formation as revealed by a toeprint assay (Figure 1B, lanes 1–3, 5). Generally, ribosomal complexes yield toeprint signals at the position +16 to +18 nt downstream of mRNA triplet in the P site of the 40S subunits. Notably, the removal of DHX29 from the reaction did not change the efficiency of 48S IC formation (Figure 1B, lane 4). After the next fractionation step, none of fractions exhibited the activity and only the combination of two split fractions reinstated the stimulatory effect (Figure 1C, left panel). Each contributing fraction included the single protein whose fractionation scheme exactly matched the purification scheme of native eIF5 and eIF5B from RRL (Figure 1C, right panel). Native eIF5 and eIF5B synergistically but not individually promoted 48S IC formation on Stem1 mRNA with the same efficiency as the preceding phosphocellulose fraction (Figure 1D, lanes 1–6). We did not observe the effect in the presence of GMPPNP (non-hydrolysable analog of GTP) suggesting that GTP hydrolysis is essential for the stimulation (Figure 1D, lanes 7 and 8). Then, we tested the effect of native eIF5 and eIF5B in the system with the full set of initiation factors including eIFs 4A, 4B and 4F. Although the presence of eIFs 4A, 4B and 4F increased the yield of 48S IC on Stem1 mRNA (Figure 1E, lanes 1 and 2), native eIF5 and eIF5B again together but not alone improved the 48S IC formation efficiency (Figure 1E, lanes 3–5). Therefore, the mechanism of stimulation by eIF5 and eIF5B is GTP-dependent and not related to the unwinding of the 5′-UTR of mRNA.


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

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

eIF5 and eIF5B are essential for stimulation of 48S IC formation. (A) Structure of Stem1 mRNA. (B, D and E) Toeprint analysis of 48S IC assembly on Stem1 mRNA in the presence of (B) RRL fraction or (D and E) different combinations of eIF5 and eIF5B. Initiation codon and position of assembled 48S IC are indicated. Lanes C/T/A/G depict corresponding DNA sequence. Toeprint assays are supplemented with quantification which shows yield of 48S IC calculated as percentage of toeprint signal to summarized signal in lane. Number of replicated experiments for quantification is at least three (n ≥ 3) and standard deviation is less than 14% (SD < 14%). (C) Left panel: purification scheme for eIF5 and eIF5B; right panel: purified eIF5 and eIF5B resolved by SDS-PAGE.
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Figure 1: eIF5 and eIF5B are essential for stimulation of 48S IC formation. (A) Structure of Stem1 mRNA. (B, D and E) Toeprint analysis of 48S IC assembly on Stem1 mRNA in the presence of (B) RRL fraction or (D and E) different combinations of eIF5 and eIF5B. Initiation codon and position of assembled 48S IC are indicated. Lanes C/T/A/G depict corresponding DNA sequence. Toeprint assays are supplemented with quantification which shows yield of 48S IC calculated as percentage of toeprint signal to summarized signal in lane. Number of replicated experiments for quantification is at least three (n ≥ 3) and standard deviation is less than 14% (SD < 14%). (C) Left panel: purification scheme for eIF5 and eIF5B; right panel: purified eIF5 and eIF5B resolved by SDS-PAGE.
Mentions: The reconstituted system comprising eIFs 1, 1A, 2, 3, 40S ribosomal subunits, Met-tRNAiMet and DHX29 promotes a 48S IC formation on the model Stem1 mRNA consisting of single-stranded 5′-UTR with centrally introduced stem (ΔG = −5.5 kcal/mol) linked with β-glucuronidase open reading frame (ORF) (Figure 1A). This mRNA has been already described and imitates the one with the moderately structured 5′-UTR (19). Despite the presence of DHX29 helicase, the efficiency of 48S IC assembly in the reconstituted system on mRNAs with structured 5′-UTR is still much lower than their translation level in the RRL (19; data not shown). Therefore, to identify the activities improving the 48S yield on Stem1 mRNA, we fractionated RRL and tested fractions in the system. After several fractionation steps, we had a phosphocellulose (P-11) fraction with a dozen of proteins which stimulated 48S IC formation as revealed by a toeprint assay (Figure 1B, lanes 1–3, 5). Generally, ribosomal complexes yield toeprint signals at the position +16 to +18 nt downstream of mRNA triplet in the P site of the 40S subunits. Notably, the removal of DHX29 from the reaction did not change the efficiency of 48S IC formation (Figure 1B, lane 4). After the next fractionation step, none of fractions exhibited the activity and only the combination of two split fractions reinstated the stimulatory effect (Figure 1C, left panel). Each contributing fraction included the single protein whose fractionation scheme exactly matched the purification scheme of native eIF5 and eIF5B from RRL (Figure 1C, right panel). Native eIF5 and eIF5B synergistically but not individually promoted 48S IC formation on Stem1 mRNA with the same efficiency as the preceding phosphocellulose fraction (Figure 1D, lanes 1–6). We did not observe the effect in the presence of GMPPNP (non-hydrolysable analog of GTP) suggesting that GTP hydrolysis is essential for the stimulation (Figure 1D, lanes 7 and 8). Then, we tested the effect of native eIF5 and eIF5B in the system with the full set of initiation factors including eIFs 4A, 4B and 4F. Although the presence of eIFs 4A, 4B and 4F increased the yield of 48S IC on Stem1 mRNA (Figure 1E, lanes 1 and 2), native eIF5 and eIF5B again together but not alone improved the 48S IC formation efficiency (Figure 1E, lanes 3–5). Therefore, the mechanism of stimulation by eIF5 and eIF5B is GTP-dependent and not related to the unwinding of the 5′-UTR of mRNA.

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