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. 2013 Jul 11;51(1):35-45.
doi: 10.1016/j.molcel.2013.04.021. Epub 2013 May 30.

eIF5A promotes translation of polyproline motifs

Erik Gutierrez  1 , Byung-Sik Shin, Christopher J Woolstenhulme, Joo-Ran Kim, Preeti Saini, Allen R Buskirk, Thomas E Dever
Affiliations

eIF5A promotes translation of polyproline motifs

Erik Gutierrez et al. Mol Cell. .

Abstract

Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate Met-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis remains elusive. We use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide bond formation between consecutive Pro residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive Pro residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates like Pro.

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Figures

Figure 1
Figure 1. eIF5A Stimulates Translation of Polyproline Motifs in vivo
(A) Schematic of Renilla-firefly luciferase reporter construct. Codon repeats were inserted in-frame between the Renilla and firefly luciferase ORFs (Letzring et al., 2010). (B) Dual luciferase reporter constructs containing 10 repeats of the indicated codon were introduced into isogenic yeast strains expressing wild type eIF5A or temperature-sensitive eIF5A-S149P. (Top panel) Following growth at semi-permissive 33°C, the firefly-to-Renilla luciferase ratio for each construct was normalized to the ratio obtained from reporters with no insert between the ORFs. (Bottom panel) The fold difference in luciferase ratios between cells expressing wild-type eIF5A and eIF5A-S149P was quantitated and then normalized to the values obtained from the no insert control. *Statistical significance for ProCCA(10) was measured by student’s t-test with a p-value <0.05. Error bars were calculated as propagated standard deviations (SD) for three independent transformants.
Figure 2
Figure 2. Translation of Three or More Consecutive Proline Codons Reveals eIF5A Dependency
Dual luciferase reporters containing 1, 2, 3, 4, 6, 8, or 10 consecutive PheUUC (F) or ProCCA (P) codons were assayed in wild type or eIF5A-S149P mutant strains and the fold differences in luciferase ratios were quantitated and normalized to the no insert control as described in Fig. 1 (error bars = propagated SD).
Figure 3
Figure 3. Expression of Polyproline-containing Proteins Requires eIF5A in vivo.
(A) Plasmids expressing HA-tagged forms of the yeast proteins LDB17, EAP1, VRP1 or eIF1A under the control of the yeast GAL1 promoter were introduced into isogenic strains expressing wild-type eIF5A or eIF5A-S149P. Cells were grown at semi-permissive 33°C in galactose medium, broken with glass beads in the presence of 10% trichloroacetic acid (TCA), and two different amounts of each extract differing by a factor of 2 were loaded in successive lanes and subject to immunoblot analysis using monoclonal anti-HA or polyclonal anti-yeast eIF2α antiserum. (B) The experiment in panel A was repeated using an LDB17 construct in which Ala codons were substituted for the nine Pro codons in the polyproline motif.
Figure 4
Figure 4. eIF5A Stimulates Synthesis of Polyproline Peptides
(A) Scheme for in vitro reconstituted translation elongation assay. (B) Fractions of MF, MFF, MFFF (left column) or MPK, MPPK, and MPPPK (right) synthesis in elongation assays (Fig. S3D–E) performed in the absence (open symbols) or presence of eIF5A (closed symbols) were plotted and fit to a single exponential equation. (C) Summary of maximum fractions of peptide synthesis (Ymax, top) and fold stimulation of Ymax by adding eIF5A (bottom) calculated from the data in panel B. Error bars are (upper) SD from at least three independent experiments and (lower) calculated propagated SD. (D) Effect of eIF5A hypusine modification on peptide synthesis. Fraction of MPPPK synthesis (Fig. S3F) in reactions lacking eIF5A, containing unmodified eIF5A (no hypusine), or containing hypusinated eIF5A prepared from E. coli (+5A, see Experimental Procedures) or purified from yeast (+5A, yeast) was plotted and fit to a single exponential equation.
Figure 5
Figure 5. eIF5A Prevents Ribosome Stalling on Consecutive Proline Codons
Reconstituted peptide synthesis assays were performed in the absence or presence of eIF5A using mRNAs encoding the peptides MFFFFF (left panel) or MPPPPP (right panel). The position of the 80S ribosome was determined by reverse transcription of the mRNA template using a [32P]-labeled primer, and C and T sequencing reactions were run alongside. Reactions lacking elongation factors were performed to identify 80S initiation complexes (IC) on the AUG codon (lanes 3 and 8). The identity of the 80S toeprint signals is indicated on the right; and the sequences of the mRNA and the corresponding amino acids are shown on the left with the sites of ribosome stalls at the 2nd and 3rd proline codons boxed.
Figure 6
Figure 6. Directed Hydroxyl Radical Probing of eIF5A Binding to 80S Ribosomal Complexes
(A) Ribbon representation of T. thermophilus EFP (right panel, pdb 3HUW, (Blaha et al., 2009)) and yeast eIF5A (left panel, pdb 3ER0) showing the protein domains (Roman numerals), the positions of the C23A and C39T mutations (black dots) that removed the native Cys residues in eIF5A, and the sites (Spheres representation) of Cys mutations for tethering Fe(II): Ser36 (green), Lys48 (magenta), Met105 (blue) and Thr126 (red). (B) Scheme for directed hydroxyl radical cleavage by Fe(II)-BABE modified forms of eIF5A in 80S complexes. (C) Directed hydroxyl radical cleavage of Met-[32P]tRNAiMet by Fe(II)-BABE- derivatized eIF5A in 80S complexes. Cleavage products were resolved on 10% (w/v) denatured polyacrylamide gels, and cleavage sites on [32P]tRNAiMet were determined by comparison to samples containing eIF5A-ΔC [WT(CysΔ), lane 8]. The tRNA ladders were prepared by digesting Met-[32P]tRNAiMet with RNase T1 (cleaves 3′ of G residue) or by base cleavage (lane 2). The tRNA residue numbers are shown at the left, and cleavage fragments are boxed. (D) Primer extension analysis of 25S rRNA cleavage fragments produced by Fe(II)-tethered to the indicated positions in eIF5A. U, C: 25S rRNA sequencing reactions using reverse transcriptase and dideoxynucleotides ddATP and ddGTP, respectively. 25S rRNA helices and the position of the L1 stalk are indicated on the left. (E) Sites of eIF5A-Fe(II)-BABE cleavages are shown on the secondary (left) and three- dimensional (pdb 1YFG, (Basavappa and Sigler, 1991)) structures of tRNAiMet. Cleavage sites are color-coded according to the site where Fe(II) was tethered on eIF5A (see panel A). (F) Summary of 25S rRNA cleavages by eIF5A-Fe(II)-BABE derivatives.
Figure 7
Figure 7. Models of 60S–Met-tRNAiMet–eIF5A Complex and eIF5A Stimulating Polyproline Synthesis
(A) Docking model of a surface representation of yeast eIF5A (orange, pdb 3ER0) and ribbons representation of tRNAiMet (cyan, pdb 1YFG, (Basavappa and Sigler, 1991)) on the ribbons structure of the yeast 60S ribosome (pdb 3O58, (Ben-Shem et al., 2010)) as viewed from the subunit interface. The position of tRNAiMet was modeled by alignment with P-site tRNA on the bacterial ribosome (pdb 2J00, (Selmer et al., 2006)), and eIF5A was docked on the 60S subunit according to the cleavage data for Met-tRNAiMet and 25S rRNA. Cleavage sites in 25S rRNA and tRNAiMet are color-coded according to the sites of Fe(II) attachment on eIF5A (see Fig. 6A). Positions of L1 stalk, 5S rRNA (black), and GTPase activating center (GAC) Stalk on the 60S subunit are indicated. (B) Magnified view of docked eIF5A and P-site tRNAiMet structure as shown in panel A (left) and rotated 180° (right). Lys51, the site of hypusine modification, is colored black. (C) Magnified view of docked eIF5A and P-site tRNAiMet (from A) overlaid on the structure of EFP (blue) from the EFP–70S structure (pdb 3HUW, (Blaha et al., 2009)) oriented as shown in panel A (left) and rotated 90° (right). (D) Model of ribosome stalled on polyproline sequence with di-proline attached to the P-site tRNA and Pro-tRNAPro in the A site (left). (Right) Binding of eIF5A near the E site places the hypusine side chain (Lys51, black) adjacent to the peptidyl-tRNA in the peptidyl transferase center (PTC) of the ribosome where it can help promote peptide bond formation with the amino acid attached to the A-site tRNA.
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