Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Abstract

Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.

Keywords: TASEP; protein charge; ribosomal proteins; translation; yeast.

Fig. 1.

Predicted and observed relationships in gene expression in the BY4741 yeast strain. (A) Illustration of the classical totally asymmetric exclusion process (TASEP) with constant rates of initiation, elongation, and termination. (B) Relationship between protein abundance (ref. 18) and the density of RPFs on the ORF, or the mRNA abundance. (C) Relationship between the number of RPFs mapped to individual mRNAs and the corresponding ORF length, mRNA level, and both. p and s are Pearson’s and Spearman’s correlation coefficients, respectively.

Fig. 2.

Analysis of protein synthesis rates. (A) Ribosome densities derived from the sequencing of RPFs (x axis) or estimated based on the relative abundance of RNAs across polysomal fractions in ref. (y axis). (B) Protein synthesis rates s can be estimated from the dynamics of light peptide (P) accumulation within a short time interval t (in minutes) after medium change (Inset). Examples of linear fits to the peptide accumulation curves for the proteins indicated in the legend. (C) Histogram of R² values of the linear fit for all 1,616 measured proteins. (D) Relationship between ribosome allocation per codon and the protein synthesis rate. Highlighted in the red box are the proteins with highest synthesis rates. The orange box highlights the cluster of RPs. p and s are Pearson’s and Spearman’s correlation coefficients, respectively.

Fig. 3.

Predicted and observed relationship between the protein synthesis rate and the ribosome density on the corresponding ORF. (A) TASEP model predictions with isoclines corresponding to individual translation initiation (gray dotted lines; rate range, 0.01 to 1.9/s; increments of 0.1 starting from second line at 0.1; mean, 0.04/s) and elongation rates (colored lines; rate range, 1 to 20 aa/s). Superimposed is the first principal component of the experimental data shown in B, for which the mean initiation and elongation rates were 0.04/s and 2.63 aa/s, respectively. (B) Similar visualization of experimental results: protein synthesis rates were measured by pSILAC and converted to molecules per mRNA per second from the expected protein mass doubling time; ribosomes densities were obtained from the fit of ribosome footprint densities to numbers of ribosomes per codons (rpc) estimated by ref. . The black contour indicates 90% of the empirical distribution approximated through the 2D kernel density estimation from the R package “MASS.”

Fig. 4.

Determinants of translation elongation rate in yeast. (A) Correlation coefficients (P values indicated on the bars) of SDR with features related to codon speed, biochemical properties of the encoded protein, and RNA secondary structure accessibility. (B) Correlation coefficients of the average accessibility of regions in the ORF of length indicated by the x axis with SDR. (C) Correlation coefficients of SDR with the probability of regions of 20 nt starting at the position indicated on the x axis relative to the A site to be in single-stranded conformation. Positions where the correlation coefficients are highly significant (P value of 0) are marked by asterisk. In all panels, Spearman and Pearson correlation coefficients are shown in blue and orange, respectively.

Fig. 5.

Influence of encoded amino acids on the translation elongation rate. (A) Positively charged proteins have low synthesis rate for the density of ribosomes on their corresponding ORFs. Each point represents an mRNA, with x and y coordinates corresponding to the ribosome density and protein synthesis rate, respectively, both on a log₁₀ scale. The color indicates the isoelectric point of the encoded protein, red indicating proteins with high pI (positively charged) and blue indicating proteins with low pI (negatively charged). (B) SDR distributions for increasing isoelectric point quantiles (left–right bins, t test, P = 2e-8). (C) Spearman (dark shade) and Pearson (light shade) correlation coefficients of SDR with amino acid frequencies in the encoded proteins (Top; only values of P < 0.05 are shown) and respective amino acids sizes (Bottom).

Fig. 6.

Properties of RPs (n = 122) compared with all other quantified yeast proteins (n = 992). Box plots of (A) tRNA adaptation index of individual ORFs and (B) isoelectric point for corresponding ribosomal and all other yeast proteins. (C) Protein synthesis rate [log₁₀(peptides/s/mRNA)] as a function of ribosome density [log₁₀(RPKM/TPM)] for transcripts encoding RPs (brown) and all other proteins (gray).

Fig. 7.

Translation parameters in yeast strains with deletions in RP genes, Δrpl6a and Δrpl7a. (A and B) Ribosome density–protein synthesis rate plots for the 2 strains; highlighted in red are outliers (having ribosome density of <0.1 RPKM/TPM; Dataset S7) in the Δrpl7a strain. (C and D) Change in the ribosome density vs. change in the synthesis rate in the Δrpl6a and Δrpl7a strains compared with wild type. The Insets show the number of transcripts in each of the 4 quadrants of the plots. In the violin plots, the distributions of density and synthesis rate changes are shown for 5 bins of SDR values (20% of transcripts in each bin), from the lowest SDR (left-most bin) to the highest (right-most bin). P values of the t test comparing the mean density and synthesis rate changes between 20% transcripts with highest and lowest SDR values, respectively, are shown.