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. 2017 Mar 16;13(3):e1006676.
doi: 10.1371/journal.pgen.1006676. eCollection 2017 Mar.

Global analysis of translation termination in E. coli

Natalie E Baggett  1 , Yan Zhang  1 , Carol A Gross  1   2   3
Affiliations

Global analysis of translation termination in E. coli

Natalie E Baggett et al. PLoS Genet. .

Abstract

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Growth defect and cold sensitivity rescue of RF mutants.
(A) Growth curves of MG1655 K-12 RF2K-12, K-12 RF2K-12ΔRF3, K-12 RF2B, K-12 RF2BΔRF3 growing in MOPS-complete glucose medium at 37°C. Doubling times are indicated. (B) Spot dilutions of cultures growing exponentially at 37°C in LB were spotted onto LB-agar plates and incubated at 37°C, 20°C, and 15°C. These indicate that the slow growth of K-12 RF2K-12ΔRF3 at 20°C is rescued in K-12 RF2BΔRF3. (C) After growth to mid-exponential phase in liquid LB cultures serial dilutions of each culture were plated on LB-agar in triplicate. Colony forming units (CFUs) of each strain were calculated for each temperature and plotted relative to the CFU of that strain at 37°C. Error bars are the standard error calculated between replicates (See Methods).
Fig 2
Fig 2. Ribosome occupancy over stop codons increases in the absence of RF3 in E.coli K-12.
Metagene analysis of ribosome footprint density in the region surrounding stop codons. The approximately 1200 well-expressed genes were aligned at their stop codons and the median normalized ribosome density at each position was calculated from ribosome profiling data of strains grown in MOPS complete-glucose media at 37°C. (A and B) Median normalized density (solid line) with standard error (shaded region) across repeat experiments. (A) Ribosome stop codon density for K-12 RF2K-12ΔRF3 (2 replicates) versus K-12 RF2K-12 (4 replicates). (B) Ribosome stop codon density for K-12 RF2BΔRF3 (5 replicates) versus K-12 RF2B (2 replicates). (C and D) Average normalized density across replicates at UAA stops (947 genes) and UGA stops (231 genes).
Fig 3
Fig 3. Increased rate of programmed frameshifting at the prfB locus.
The translation of prfB is regulated through a programmed +1 frameshift at codon 26, shown as a UGA frameshift (dotted line). Successful frameshifting results in the complete RF2 protein. Ribosome profiling data was used to estimate frameshifting over the internal UGA stop codon by comparing the ribosome occupancy (RPKM) across the first small ORF prior to the UGA stop and second ORF encoding full-length RF2. This ratio gives a rough estimate of percent frameshift at the prfB locus for each RF mutant.
Fig 4
Fig 4. Genome wide increase in post-ORF occupancy in K-12 ΔRF3.
(A) Ribosome occupancy in the region past the annotated stop codon was calculated for all genes that had an intergenic region of 65 bases or greater, measured from the stop codon of the upstream gene of interest to the start codon of the downstream gene, and met expression thresholds for both mRNA abundance and ribosome footprints (1656 genes for the deepest sequenced library). The metric relative post-ORF ribosome occupancy (RPOR) was calculated from the average occupancy over the annotated ORF and the average occupancy in a post-ORF window 20-60bp after the stop codon. By using the average occupancy for both the post-ORF and ORF, we reduce the impact of length in these calculations. (B) The distribution of RPOR values from 0 to 2.0 for K-12 RF2K-12, K-12 RF2K-12ΔRF3, and K-12 RF2BΔRF3 is shown as a cumulative distribution function for one experiment with 1139 genes after all zero RPOR values were removed. A shift towards higher RPOR values in the K-12 RF2K-12ΔRF3 strain relative to the K-12 RF2K-12 strain is statistically significant (p-value 0.04; Kolmogorov-Smirnov (K-S) test), but the small shift between the K-12 RF2BΔRF3 and K-12 RF2K-12 strains is not (p-value 0.84, K-S test). (C) A histogram of the distribution of RPOR values ≥0.2 for K-12 RF2K-12 and K-12 RF2K-12ΔRF3 compares the number of genes in each range of RPOR for both strains.
Fig 5
Fig 5. Schematic explaining ORF classification as recoding or non-recoding.
Genes with high relative post-ORF ribosome occupancy (RPOR) were individually analyzed to determine the origin of ribosomes in the post-ORF region. Left hand panel: Schematic of ribosome footprints for an annotated gene (shaded light green) and its post-ORF region (shaded red stripes). The ORF start (green triangle) and stop (red triangle) codons are indicated. Right hand panel: a zoomed-in post-ORF region with all possible start (green line) and stop (red line) codons in each reading frame indicated. Upper right hand panel: A post-ORF region where ribosome density abruptly decreases after the stop codon in the -1 frame, classified as a putative recoding event. The hypothesized extended region is shaded in red and the putative stop codon is marked with a red triangle. Lower right hand panel: A post-ORF region where ribosome density does not decrease after any possible the stop codon, classified as a non-recoding event.
Fig 6
Fig 6. ΔRF3 increases recoding in genes identified with high ribosome occupancy post-ORF.
(A and C) Left panels: Normalized ribosome footprints are shown across nudL (A) or panZ (C). The ORFs (shaded green) and post-ORF region, with hypothesized extensions (shaded red) are indicated. Right panels: zoomed-in post-ORF region with all possible start (green line) and stop (red line) codons in each reading frame indicated. The stop codon of the C-terminal streptavidin tagged extension is indicated with a red triangle within the shaded red extension. (B and D) Western blots of strains containing N-terminally FLAG-tagged and C-terminally streptavadin tagged NudL (B) or PanZ (D) and using SurA as a loading control (lc). The NudL C-terminal streptavidin tag is in the 0 frame, and that of PanZ is in the -1 frame. (A) The post-ORF region of nudL has reduction in ribosome occupancy correlated with stop codons in both the 0 and -1 frame. We determined the 0 frame produces an extended NudL product (S7 Fig). (B) Blotting of α-FLAG indicated the NudL protein, 22.78kDa, and the readthrough extension product at 24.06kDa which is also seen in the α-streptavidin blot. (C) The post-ORF region of panZ reveals several possibly productive stop codons. (D) Blotting of α-FLAG indicated the PanZ protein, 15.84kDa, and the -1 frame extension product at 20kDa, which is also seen in the α-streptavidin blot.
Fig 7
Fig 7. ΔRF3 reduces expression of biosynthetic genes controlled by leader peptide attenuation.
(A and B) Scatter plots of ribosome footprint density in RPKM for K-12 RF2K-12 and K-12 RF2K-12ΔRF3 strains for all genes above minimum read threshold, with biosynthetic genes under the control of leader peptide attenuation in red are all others in grey. (A) Rich media samples of K-12 RF2K-12 and K-12 RF2K-12ΔRF3 grown in MOPS-complete glucose medium are averaged data across multiple replicates. (B) Minimal medium ribosome footprint densities between K-12 RF2K-12 and K-12 RF2K-12ΔRF3 in MOPS minimal glucose medium are plotted for a single replicate. (C) Schematic of the chromosomally integrated reporter constructs measuring expression of the leader peptide (reporter #1) or downstream gene expression (reporter #2), both under control of their native promoter. (D) Operon expression in K-12 RF2K-12 and K-12 RF2K-12ΔRF3 strains was calculated by dividing the β-galactosidase activity of reporter #2 by activity of reporter #1 and normalizing to the K-12 RF2K-12 strain in in MOPS complete-glucose rich media. (E) Increased attenuation in K-12 RF2K-12ΔRF3 strain may result from increased occupancy over the stop codon and post-ORF region. This mechanism would stabilize the formation of the downstream terminator loop over the anti-terminator loop.
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