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

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.


Aminoacylation of tRNA
In vitro transcribed tRNA i Met and rabbit native total tRNAs (Promega) were aminoacylated with methionine in the presence of recombinant E.coli methionyl-tRNA synthetase as described (19). For methionyl-puromycin assay, in vitro transcribed tRNA Met i was aminoacylated using 35Slabelled methionine (5 x 10 5 cpm/pmol).

eIF5 and eIF5B purification
Native eIF5 and eIF5B were purified from the 40-50% ammonium sulfate (AS) precipitation fraction of the 0.5 M KCl ribosomal salt wash (RSW) that was prepared from 450 ml of RRL. The fraction was dialyzed against buffer B (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 2 mM DTT, 10% glycerol) + 100 mM KCl and applied to a diethylaminoethyl cellulose (DEAE) column equilibrated with buffer B + 100 mM KCl. Both proteins were eluted at 100-250 mM KCl. This fraction was dialyzed against buffer B + 100 mM KCl and applied to a phosphocellulose (P-11) column equilibrated with buffer B + 100 mM KCl. eIF5 and eIF5B were eluted at 400-800 mM KCl, dialyzed against buffer B + 100 mM KCl and applied to a FPLC MonoQ 5/50 GL column. Fractions were collected across a 100-500 mM KCl gradient. eIF5 eluted at ~360 mM KCl and eIF5B-at 480mM KCl.
Recombinant eIF5B was expressed in 1 L of E.coli BL21(DE3) after induction by 0.1 mM IPTG for 16 h at 16 o C and isolated by affinity chromatography on Ni-NTA agarose followed by FPLC on MonoQ column. Fractions were collected across a 30-500 mM KCl gradient. eIF5B elutes at 100mM KCl.

Purification of eIF1A and eIF5B mutants
Recombinant eIF1A I144A, eIF5B T665A and eIF5B H14 were expressed in 1 L of E.coli BL21(DE3) after induction by 0.1 mM IPTG for 16 h at 16 o C and isolated by affinity chromatography on Ni-NTA agarose followed by FPLC on MonoQ column. Fractions were collected across a 100-500 mM KCl gradient for eIF1A mutant and a 30-500 mM KCl gradient for eIF5B mutants. eIF1A I144A, eIF5B T665A and eIF5B H14 elute at 310 mM, 70 mM and 100 mM KCl, respectively.

Toeprint assay
We estimated the yield of 48S IC assembled on different mRNAs and viral RNAs employing toeprint assay as described (19). This assay involves the extension by the reverse transcriptase of the radiolabelled primer annealed to the mRNA. Generally, ribosomal complexes yield toeprint signals at the leading edge of the 40S subunits at the position +16-+18 nt downstream of mRNA triplet in the P site. Signals are visualized after the electrophoresis of cDNA in the polyacrylamide gel and autoradiography. containing buffer A with/without 10 pmol eIFs 1/1A/5/5B, 5pmol eIF3, 1.75 pmol 40S subunits, 2.5 pmol 80S ribosomes, and 10 pmol (CUUU) 9 RNA for 15 min at 37 o C, and subjected to PEI cellulose chromatography as described (19).

Purification and analysis of ribosomal complexes by sucrose density gradient centrifugation
For GTPase assay, 43S PIC assembled in the presence of 0.33M [-32P]GTP in a 400 l reaction mixture with scaled amounts of initiation components was purified by centrifugation through 10-30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 75 min.
To examine the effect of eIF5 and eIF5B on the composition of 43S PIC and 48S IC, ribosomal complexes reconstituted in a 400 l reaction mixture with scaled amounts of initiation components were subjected to centrifugation through 10-30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 75 min. Fractions that corresponded to ribosomal complexes were resolved in SDS-PAGE and stained with Sypro Ruby Red (Invitrogen) or assayed by immunobloting with antibodies against eIF2 (Abcam).

Methionyl-puromycin assay
To compare the activity of 48S IC in the formation of 80S IC, methionyl-puromycin assay was employed. 48S ICs were reconstituted on CAA-CUG mRNA with 35S-labelled Met-tRNA i Met in the presence/absence of eIFs 5/5B, supplemented with 60S subunits and eIFs 5/5B (if they were not added before), and additionally incubated for 10 min at 37 o C. After assembly, ribosomal complexes were treated with 1 mM puromycin for 40 min at 37 o C and extracted with ethyl acetate. 35S-labelled methionyl-puromycin formation was measured by scintillation counting of ethyl acetate extract.

UV cross-linking assay
To examine the effect of eIF5 and eIF5B on 43S PIC rearrangement, we performed UV crosslinking experiments. 48S ICs were reconstituted with/without eIFs 5/5B on co-transcriptionally 32P-labelled "-3U" and "+4U" mRNAs containing 4-thioU at -3 and +4 key context positions, respectively. To keep the integrity of ribosomal complexes, we omitted the purification step by SDG centrifugation. After assembly, 48S ICs were irradiated at 360 nm for 30 min on ice using a UV-Stratalinker (Stratagene) and digested with 5 units RNAse A for 10 min at 37 o C. To identify cross-linked proteins, ribosomal complexes were assayed by SDS-PAGE and autoradiography.

Analysis of eIF1A and eIF5B mutants activity in 80S IC formation
To test the activity of mutants, 80S ICs were assembled on 32P-labelled -globin mRNA with eIF1A I144A, eIF5B T665A, eIF5B H14 or their wild-type forms in a 400 l reaction mixture with scaled amounts of initiation components. Next, ribosomal complexes were resolved by centrifugation through 10-30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 105 min. To assign ribosomal peaks, optical density of gradient fractions was measured at 260 nm. The yield of 48S IC and 80S IC was determined by Cherenkov counting of