1. Volume 59, Issue 5, Pages 744-754 (September 2015)
Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co- translational Protein Folding 
Chien-Hung Yu, Yunkun Dang, Zhipeng Zhou, Cheng Wu, Fangzhou Zhao, Matthew S. Sachs, Yi Liu 
Molecular Cell 
Volume 59, Issue 5, Pages (September 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 744-754DOI: (10.1016/j.molcel.2015.07.018)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Codon Usage Affects Translation Elongation Rate
(A) Real-time measurement of firefly luciferase (Luc) activity using the Neurospora cell-free translation system at 26°C. Recorded relative light units (RLUs) were plotted versus translation reaction time in 30-s intervals. TFAs are indicated by arrows. Data are represented as mean ± SD.
(B) SDS-PAGE analysis showing the [35S]Met-labeled translational products from WT and OPT Luc mRNAs obtained from micrococcal nuclease-treated Neurospora cell-free lysates. NC is the background translation (without added mRNA). The arrow indicates the position of full-length LUC.
(C and D) Scatter plots showing the effect of codon usage frequency on elongation speed by comparison the TFA of the indicated Luc mRNAs. (C) N-OP-WT and M-OP-WT have codons corresponding to aa 2–223 and 224–423 optimized, respectively. (D) N-WT-OPT and M-WT-OPT have codons corresponding to aa 2–223 and 224–423 as WT sequences, respectively. Means indicated by horizontal bars for all measurements were derived from 4–10 independent experiments. ∗p < 0.05, ∗∗p < 0.01.
(E) Plot showing the real-time LUC activity of FRQ-WT Luc fusion proteins. The TFA of each construct is indicated by an arrow. Data are represented as mean ± SD.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Elongation Rate Is Affected by Codon Usage Frequency Rather Than by Codon-tRNA Balance or mRNA Secondary Structures
(A) Scatter plot showing the TFA of WT and OPT Luc using Neurospora cell-free lysates at different temperatures.
(B) Scatter plot showing the TFA of WT and OPT Luc in micrococcal nuclease-treated and untreated Neurospora cell-free lysates.
(C) Scatter plot showing the TFAs of programmed translation reactions using micrococcal nuclease-treated Neurospora lysates with indicated concentrations of WT and OPT Luc mRNAs.
(D) Plot showing the real-time Luc activity of WT and OPT mRNAs using nuclease-treated yeast cell-free extracts. The TFAs are indicated by arrows.
Means are indicated by horizontal bars and were derived from 3–6 independent experiments. Data are represented as mean ± SD.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Codon Usage Affects Local Ribosome Traffic on mRNA
(A) SDS-PAGE analysis of [35S]Met-labeled total translation products and isolated RNCs of WT and OPT Luc after 12 min of translation reaction. The reaction was terminated by cycloheximide (0.5 mg/ml, final concentration).
(B) SDS-PAGE analysis of [35S]Met-labeled translation products of WT and OPT Luc after 12 min at indicated temperatures. The full-length Luc protein is indicated by an arrow.
(C) SDS-PAGE analysis of [35S]Met-labeled translation products of the indicated Luc mRNAs. The bar on the left of each lane demonstrates the codon usage patterns of the Luc gene: black indicates optimized codons, and gray indicates WT codons.
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6. Figure 4
Ribosome Profiling Analyses Reveal that Ribosome Occupancy on mRNA Negatively Correlates with Codon Usage Frequency In Vitro and In Vivo
(A) RPF profile on Luc mRNAs translated in vitro. The sequenced RPFs of indicated Luc constructs from Neurospora in vitro translation are mapped to the corresponding mRNAs. The average CAI values of each gene fragment are indicated.
(B) RPF profile on Luc mRNAs translated in vivo. The RPFs of indicated ccg-1-driven Luc constructs, which are transformed into the his-3 locus of the Neurospora genome, were mapped to the corresponding Luc gene.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Genome-wide Negative Correlations between RPFs and Synonymous Codon Usage In Vivo
Scatter plot showing the negative correlation between the RCDR and the RCUF for all coding genes. Spearman’s rank correlation coefficient (ρ) and the associated p value are indicated.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Codon Usage Regulates Protein Activity by Affecting Co-translational Protein Folding
(A) Scatter plot showing the comparison of WT and OPT LUC-specific activities in the Neurospora cell-free lysate.
(B) Scatter plot showing the ratio of relative LUC activity between OPT and WT mRNA after 12 min of in vitro translation at indicated temperatures.
(C) Top panel: SDS-PAGE analysis showing the levels of WT and OPT LUC at the indicated time points after the addition of trypsin (20 μg/ml). Lower panel: densitometric analyses of LUC levels from four independent experiments. Means are indicated by horizontal bars and were derived from 3–4 independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Codon Usage in a Defined Structural Region Affects LUC Activity In Vitro and In Vivo
(A) Top panel: diagrams showing the codon-optimized region (black) of each WT-based Luc construct. Bottom panel: scatter plot showing relative specific LUC activities of in vitro translated and indicated Luc mRNAs.
(B) Top panel: diagrams indicating the regions in which codons are in WT sequence (gray) in otherwise OPT Luc constructs. Bottom panel: scatter plot showing the comparison of the specific Luc activities of in vitro translated Luc mRNAs.
(C) Top panel: diagrams showing the regions in which codons are optimized (black) in WT Luc. Bottom panel: the relative specific Luc activities of indicated Luc constructs.
(D) Scatter plot showing the comparison of LUC specific activity in vivo. The frq promoter-driven OPT Luc and (172–223) WT-Luc were transformed into Neurospora at the his-3 locus. The 24-hr germinating conidia were harvested, followed by LUC activity (RLU) measurement and LUC protein quantification by western blot. The RLU and LUC protein levels were normalized to the value of one of the OPT samples. The specific Luc activities were calculated by dividing the normalized RLU by the normalized Luc protein level.
Means are indicated by horizontal bars and were derived from 3–5 independent experiments. ∗p < 0.05, ∗∗p < See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions