Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria
- Fred Hutchinson Cancer Research Center, United States
Figures
Figure 1
Effect of ribosome stalling on measured protein level, mRNA level, and polysome occupancy.
(A) Schematic of ribosome stalling reporters used in B–E. Blue vertical lines show the location of CTA Leu codons that cause ribosome stalling during Leu starvation in E coli. Locations of CTG Leu …
Figure 2
Computational modeling of ribosome kinetics at stall sites.
(A) Distinct ribosome states that were considered during computational modeling of each elongation cycle. (B) Schematic of the three kinetic models of stalled ribosomes considered in this work. The …
Figure 3
Distinct predictions from three kinetic models.
Predicted effect on protein synthesis rate upon varying the initiation rate (A), the number of ribosome stall sites (B), and the distance between two stall sites (C) of a yfp mRNA. The schematics …
Figure 4 with 1 supplement
Predicted vs. measured YFP synthesis rates during Leu starvation upon variation in initiation rate.
yfp reporters with a single CTA codon and one of five initiation regions are shown in the schematic. X axis – Measured YFP synthesis rate during Leu-rich growth was used as a proxy for the …
Figure 4—figure supplement 1
Predicted and measured YFP synthesis rates during Leu starvation (A-G) or Ser starvation (H) from yfp reporters with a single stall-inducing codon and one of five initiation region variants in Figure 4 (labeled 1–5).
Each panel represents data for initiation rate variants having the indicated stall-inducing codon (Leu CTT, CTC, or CTA; Ser TCG) at one of three different Leu codon locations or one Ser codon …
Figure 5 with 1 supplement
Predicted vs. measured YFP synthesis rates during Leu starvation upon variation in number of stall sites.
yfp reporters having one to four CTA codons at the five Leu positions in yfp are shown in the schematic. X axis – location of CTA codons in each of the yfp variants. Y axis – Predicted and measured …
Figure 5—figure supplement 1
Predicted and measured YFP synthesis rates during Leu starvation (A-B) or Ser starvation (C) from yfp reporters having stall-inducing codons CTC (A), CTT (B), or TCG (C) at one or two among either five Leu positions shown in the upper schematic or six Ser positions in yfp in the lower schematic.
The Leu or Ser positions are labeled by their order of occurrence along yfp relative to the start codon (22 Leu codons, 8 Ser codons total). X axis, Y axis, RMS error % and simulation parameters are …
Figure 6 with 1 supplement
Predicted vs. measured YFP synthesis rates during Leu starvation upon variation in distance between stall sites.
yfp variants with two CTA codons are arranged by increasing value of d, the distance between the CTA codons. X axis – location of CTA codons in each of the yfp variants. Y axis – Predicted and …
Figure 6—figure supplement 1
Predicted and measured YFP synthesis rates during Leu starvation from yfp reporters having CTC codons at one or two among five Leu positions in yfp shown in the schematic.
X axis, Y axis, RMS error % and simulation parameters are as shown in the main figure.
Figure 7
Selectivity, robustness, and ribosome density in the collision-stimulated abortive termination model.
(A) Predicted effect of varying the abortive termination rate on protein synthesis rate from a yfp mRNA having no stall site (top panel) or a single stall site (bottom panel). The TJ model …
Author response image 1
Western blot to demonstrate abortive termination at stall-inducing codons during leucine starvation
https://doi.org/10.7554/eLife.23629.019
Author response image 2
Comparison of measured YFP levels when expressed from chromo- some and plasmid during Leu-rich growth
https://doi.org/10.7554/eLife.23629.020
Author response image 3
Comparison of measured YFP levels when expressed from chromo- some and plasmid during Leu starvation
https://doi.org/10.7554/eLife.23629.021
Author response image 4
Distribution of simulation error.
Simulation error was calculated as the deviation of the YFP synthesis rate from the mean across an ensemble of 5000 identically simulations for each of the three kinetic models considered in the …
Author response image 5
Distribution of measurement error.
Measurement error was calculated as the standard error of the mean measured YFP synthesis rate for all the yfp mutants used in our manuscript (N=115). We note that even though standard error is …
Author response image 6
Distribution of modeling error.
Modeling error was calculated as the residual difference between the mean prediction for YFP synthesis rate from each of the three kinetic models and the measured YFP synthesis rate for the …
Additional files
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Supplementary file 1
Simulation parameters for Figure 3
- https://doi.org/10.7554/eLife.23629.012
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Supplementary file 2
Simulation parameters for Figure 3 (including Figure supplement)
- https://doi.org/10.7554/eLife.23629.013
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Supplementary file 3
Simulation parameters for Figure 5 (including Figure supplement)
- https://doi.org/10.7554/eLife.23629.014
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Supplementary file 4
Simulation parameters for Figure 6 (including Figure supplement)
- https://doi.org/10.7554/eLife.23629.015
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Supplementary file 5
Simulation parameters for Figure 7
- https://doi.org/10.7554/eLife.23629.016
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Supplementary file 6
Parameters common to all simulations
- https://doi.org/10.7554/eLife.23629.017
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Supplementary file 7
List of strains and plasmids
- https://doi.org/10.7554/eLife.23629.018
