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Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis - PubMed

  • ️Wed Jan 01 2014

Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis

Steven J Hersch et al. J Biol Chem. 2014.

Abstract

Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo. The α- and β-subunits of ATP synthase, AtpA and AtpD, are translated from the same mRNA transcript, and both contain a PPG motif; however, proteomic analysis revealed that AtpD levels are strongly dependent on EF-P, whereas AtpA levels are independent of EF-P. Using these model proteins, we systematically determined that EF-P dependence is strongly influenced by elements in the 5'-untranslated region of the mRNA. By mutating either the Shine-Dalgarno sequence or the start codon, we find that EF-P dependence correlates directly with the rate of translation initiation where strongly expressed proteins show the greatest dependence on EF-P. Our findings demonstrate that polyproline-induced stalls exert a net effect on protein levels only if they limit translation significantly more than initiation. This model can be generalized to explain why sequences that induce pauses in translation elongation to, for example, facilitate folding do not necessarily exact a penalty on the overall production of the protein.

Keywords: ATP Synthase; Bacterial Pathogenesis; Gene Regulation; Ribosome; Translation Elongation Factor; Translation Initiation; mRNA.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.
FIGURE 1.

AtpD but not AtpA shows decreased fluorescence in efp mutant Salmonella throughout the growth curve. Sixteen-hour growth curves of wild-type (solid lines) and efp mutant (dashed lines) Salmonella containing pXG10sf plasmids with AtpD (A and B) or AtpA (C and D) were inserted as a translational fusion to GFP. GFP fluorescence (black lines in A and C) and optical density at 600 nm (gray lines in A and C) were measured every 15 min. The ratio of GFP fluorescence/A600 is also shown (B and D). At least three independent replicates were conducted, and one representative replicate is shown. AFU, arbitrary fluorescence units.

FIGURE 2.
FIGURE 2.

The 5′-UTR plays a role in EF-P dependence in addition to residues immediately upstream of the PPG motif. A, outline of pXG10sf-AtpD and -AtpA constructs. Swap constructs are shown with regions of the atpA gene swapped into the pXG10sf-AtpD construct. The naming scheme is indicated at left for each construct. B, regions of the atpA gene were swapped into the pXG10sf-AtpD construct (as shown in A). The data show the ratio of GFP fluorescence in Δefp/WT Salmonella normalized to A600. Unaltered (wt), a PPG::PLG mutation (P214L), the “12aa” construct, and pXG10sf-LacZ have been previously published (41) and are shown for comparison. C, as in B but showing constructs with regions of the atpD gene swapped into pXG10sf-AtpA (inverse of depiction in A). The values are the means of at least three biological replicates, and the error bars show one standard deviation.

FIGURE 3.
FIGURE 3.

The 5′-UTR regions affecting EF-P dependence include the ribosome binding site and correlate with expression levels. A, sequence of the 5′-UTRs included in pXG10sf-AtpD and -AtpA constructs. Putative Shine-Dalgarno sequences are underlined. Base positions relative to the translation start site (ATG) are indicated below. B, regions of the atpA 5′-UTR were serially swapped into the corresponding position of pXG10sf-AtpD. For example, “−15–1” indicates the 15 bp from position −15 to −1 (relative to the ATG start codon) were swapped. The data show GFP fluorescence in arbitrary fluorescence units (AFU) normalized to A600 in WT (dark gray) and Δefp (light gray) Salmonella. The numbers above the columns indicate the Δefp/WT ratio. C, as in B but with regions of the atpD 5′-UTR swapped into the pXG10sf-AtpA construct. The values are the means of at least three biological replicates, and error bars show one standard deviation.

FIGURE 4.
FIGURE 4.

Immunoblot of GFP levels follow similar trends as fluorescence. Western blot probing for GFP expressed from pXG10sf constructs in WT and Δefp Salmonella grown to mid-log phase under conditions similar to those used for fluorescence measurements. GFP− control is wild-type cells expressing pXG10sf-AtpD but with a FLAG tag replacing GFP. Unless otherwise indicated, 10 μg of protein lysate was loaded for each sample. DnaK was included as a loading control. The experiment was conducted in triplicate, and one representative replicate is shown. For the “AtpD-AtpA UTR” and “AtpD AGAGG::AGACG” constructs in which fluorescence levels were less than 2-fold greater than no-plasmid controls, protein levels were very low or nondetectable in both the WT and efp mutant strains. The “AtpA-AtpD UTR from −60–46,” “AtpA-AtpD from ATG-PPG,” “AtpD-AtpA UTR and 12aa,” and “AtpD-AtpA UTR and P214L” constructs also had similarly low fluorescence levels (Figs. 2 and 3). These constructs were omitted from all downstream analyses.

FIGURE 5.
FIGURE 5.

The effect of the 5′-UTR on EF-P dependence requires an intact stall motif. A, the −45–31 region of the atpA 5′-UTR was swapped into the pXG10sf-AtpD construct in combination with mutating the PPG motif to PLG. The data show GFP fluorescence in arbitrary fluorescence units (AFU) normalized to A600 comparing expression in WT (dark gray) to Δefp (light gray) Salmonella. The numbers above the columns indicate the Δefp/WT ratio. B, as in A but with the 5′-UTR of atpD (−74–1) swapped into the pXG10sf-AtpA construct. The values are the means of at least three biological replicates, and error bars show one standard deviation.

FIGURE 6.
FIGURE 6.

Single base mutations in the SD sequence or start codon alter expression and EF-P dependence. A, mutations in the SD sequence or in the ATG start codon of pXG10sf-AtpD. The wild-type sequence (AGAGG) is underlined in Fig. 3A. The data show GFP fluorescence in arbitrary fluorescence units (AFU) normalized to A600 comparing expression in WT (dark gray) to Δefp (light gray) Salmonella. The numbers above the columns indicate the Δefp/WT ratio. Unaltered (wt) construct is shown for comparison. B, as in A but with mutation in the SD sequence of the pXG10sf-AtpA construct. The wild-type sequence (AGGGGA) is underlined in Fig. 3A. The values are the means of at least three biological replicates, and error bars show one standard deviation.

FIGURE 7.
FIGURE 7.

Translation initiation and elongation stall strength influence protein level. A, GFP fluorescence data plotted to compare expression in WT (x axis) and Δefp (y axis) Salmonella. The y axis is expanded from 0 to 1 to clarify differences at low fluorescence levels. Each point represents fluorescence data for one pXG10sf construct. The specific construct for each data point is indicated by numerical label referring to Table 1. Icon groups signify constructs that all have the same ORF (indicated in key at bottom right) and only differ from one another in the 5′-UTR. For the AtpD and AtpA ORF groups, the data points were fit to a 1 − exp curve shown in the inset at the top left. P214L and P281L groups were connected linearly, and the dashed line indicates equal fluorescence in the WT and Δefp mutant. The data are shown as arbitrary fluorescence units (AFU) normalized to A600 and are the means of at least three biological replicates. Error bars showing one standard deviation are included for both x and y axes. “AtpD-AtpA 12aa” indicates 12 codons upstream of the atpA PPG motif were swapped into the AtpD construct, “AtpA-AtpD 12aa” is the reciprocal. B, fluorescence data from E. coli containing the pBAD30XS plasmid with the indicated polyproline motifs inserted at the fourth codon of GFP. For each motif construct, four start codon mutations were generated: from highest to lowest expression in wild type: AUG (wt), GUG, UUG, AUC, and CUG. Start codon mutant constructs with the same polyproline motif were plotted as a group with a corresponding 1 − exp curve of best fit. “No motif” has no inserted motif and is included as an EF-P independent control. Shown is a fluorescence ratio of GFP normalized to mCherry expressed from the same mRNA but with its own ribosome binding site. The y axis is expanded from 0 to 2 to clarify differences at low fluorescence levels. C, computational model comparing the effect of varying rates of translation initiation and stall clearance on protein synthesis (terminating ribosomes). Black circles indicate tested combinations of stall progression and initiation frequency and are connected linearly. Details of the model are described under “Experimental Procedures.”

FIGURE 8.
FIGURE 8.

Translation pause sites accumulate ribosomes and can affect downstream ribosome occupancy. A and B, ribosome occupancies from the computational translation model described under “Experimental Procedures” with a stall between codons 99 and 100. Shown are the number of ribosomes occupying each codon for varying initiation frequencies when the stall progression frequency was set to 1/100 (0.01). Ribosome occupancies are shown at 2,000 cycles (A) or 10,000 cycles (B) of running the program. C and D, ribosome profiling data from Elgamal et al. (41) showing ribosome occupancies within the AtpD (C) and AtpA (D) ORFs. A black line indicates ribosome occupancy in wild-type E. coli, and a gray line indicates ribosome occupancy in the efp mutant. The site of the PPG motif is indicated by a vertical dotted line.

FIGURE 9.
FIGURE 9.

Peptide abundance ratios do not change significantly before and after APP, PPG, or PPP motifs in Salmonella. A, previously conducted SILAC data were analyzed comparing the mean peptide ratios (WT/Δefp) before and after the first APP, PPG, or PPP motif of the protein (29). Only proteins with an APP, PPG, or PPP motif and at least two peptides conclusively identified both before and after the motif were analyzed (40 proteins). A t test was used to calculate statistical significance comparing the average peptide abundance ratio pre- versus postmotif. Gene names and p values are shown for proteins with a difference between pre- and postmotif of greater than two. AtpD and AtpA are also indicated. The only protein with p < 0.05 is highlighted as a black square (Lon). The linear regression is shown with coefficient of determination (R2) indicated at right. A dashed line indicates a 1:1 regression. B, all Lon peptides detected in the previously conducted SILAC assay are shown plotting peptide ratio (WT/Δefp) against their location within the full-length Lon protein (29). The dashed line indicates the location of the PPG motif. The average values of all peptide ratios before or after the PPG motif are shown as a solid line, and error bars indicate one standard deviation.

FIGURE 10.
FIGURE 10.

Model of the interplay between translation initiation rate and elongation stalls. This figure depicts translation of an mRNA transcript with low initiation rate (left columns) or high initiation rate (right columns) and the subsequent effect of no elongation stall (top row), a weak stall (middle row), or a strong stall (bottom row). Black line, mRNA transcript; purple box, start codon; brown box, stop codon; blue ovals, translating ribosomes; yellow stop sign, weak stall motif; red stop sign, strong stall motif. ++++, high degree of protein synthesis per transcript; ++, medium protein synthesis per transcript; +, low level of protein synthesis per transcript.

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