Fig 1.
Eq (4) accurately determines the translation-initiation rate from simulated S. cerevisiae ribosome profiles and its application to experimental data.
(A) Translation-initiation rates determined by applying Eq (4) to simulated ribosome profiling data are plotted against the actual initiation rates used in the simulations. These initiation rates were calculated using Eq (S10) for the average protein synthesis times. (B) Same as (A) but the average protein synthesis times were measured from our simulations of the translation process. The solid lines in (A) and (B) are the lines of the best fit. (C) The distribution of in vivo translation-initiation rates measured by applying Eq (4) to experimental data involving 1,287 S. cerevisiae high coverage transcripts.
Fig 2.
Measuring average elongation rate by applying Eq (7) to in silico and in vivo ribosome run-off data.
(A) Normalized average ribosome read density (), Eq. (S15), calculated from simulated ribosome run-off experiment is plotted as a function of codon position for run-off times of 0, 5, 10, 15, 20, 25 and 30 s−1 with black, red, blue, cyan, pink, yellow and green data points, respectively. (B) The average time taken to fully deplete the normalized average ribosome read density within a window of the most 5′ codons positions in S. cerevisiae transcripts are plotted against the most 3′ codon position of the window. (C) Normalized average ribosome read density, calculated from in vivo run-off experimental data reported in Ref. [26], are plotted as a function of codon position for the run-off times of 0, 90, 120 and 150 seconds with black, red, blue and cyan data points, respectively. (D) The average time taken to fully deplete the normalized average ribosome read density within a window of the most 5′ codons positions in mouse stem cells transcripts are plotted against the most 3′ codon position of the window. The negative intercept reflects the time taken by harringtonine to engage with ribosomes.
Fig 3.
Eq (12) accurately determines codon translation times from simulated ribosome profiles.
(A) Average translation time of a codon in YER009W S. cerevisiae transcript is plotted as a function of its position within the transcript. The true codon translation times in the simulations are plotted as green boxes, blue and black data points are the translation times measured using Eq (12). Blue data points were calculated using the average protein synthesis time measured from the simulations and relative ribosome density calculated using a large number of in silico ribosome profiling reads. Black data points were calculated using the average protein synthesis time estimated from the scaling relationship (Eq. (S10)) and the relative ribosome density calculated from the in silico reads which were equal to the reads aligned to the same transcript in the experiment [16]. (B) Measured codon translation times, plotted with black and blue data points in (A), are plotted against true codon translation times in the simulations in the top and bottom panel, respectively. (C) Probability distribution of the R2 correlation between the true and calculated codon translation times for the 85 S. cerevisiae transcripts. (D) Probability distribution of the slope of the best-fit lines between the estimated and true codon translation times for the 85 S. cerevisiae transcripts. The high R2 in (C) and median slope of 1.00 in (D) indicate that Eq (12) can, in principle, accurately measure absolute rates.
Fig 4.
Translation-initiation rates measured using Eq (4) reproduce previously reported correlations with molecular properties.
In vivo translation-initiation rates of S. cerevisiae transcripts are plotted against the inverse of their CDS length, folding energy of mRNA molecule near the 5′ cap and protein copy number in (A), (B) and (C), respectively. (D) The copy number of S. cerevisiae proteins are plotted as a function of the product of the initiation rate of the transcripts that encode them and that transcript’s copy number in a cell. (E) mRNA copy number is plotted against the translation-initiation rate.
Fig 5.
Wide variability in individual codon translation rates in vivo.
(A) Probability density functions for translation times of AUU, GAC and UGG codons in Nissley dataset. Median translation times for AUU, GAC and UGG codon are 127, 208 and 331 ms, respectively. (B) The translation time profile of S. cerevisiae transcript YAL038W from Nissley dataset is shown between codon positions 150 and 450. AAG codon (colored red) is translated in 362.8 ms at the 196th codon position and in 58.6 ms at 413th codon position.
Fig 6.
Molecular factors shaping the variability of individual codon translation rates.
(A-B) Median translation times of codon types are negatively correlated with cognate tRNA abundance estimated by (A) gene copy number and (B) RNA-Seq gene expression. (C) Probability distribution of translation times of codons in the A-site either when a proline is present in the P-site (green) or when a proline is not present in the P-site (blue). (D-E) Percentage difference in median translation times when mRNA structure is present relative to when it is not present is plotted as a function of codon position after the A-site. Grey bars indicate results that are not statistically significant. Error bars are the 95% C.I. calculated using 104 bootstrap cycles; significance is assessed using the Mann-Whitney U test corrected with the Benjamini Hochberg FDR method for multiple-hypothesis correction. mRNA structure information used in (D) and (E) are provided by in vivo DMS and in vitro PARS data, respectively. (F) Scatter plot of the median translation times of pairs of codon types that are decoded by the same tRNA molecule. The red line is the identity line. The list of tRNA molecule names and decoded codon types were taken from Ref. [54]. Error bars are standard error about the median translation time calculated with 104 bootstrap cycles.