Quantification of elongation stalls and impact on gene expression in yeast

Figures

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  Fig. 1.. Assay validation via elongation rate measurements. A: Diagram of yeast-optimized constructs of various lengths. Optimized YFP (optYFP) or both optYFP and optimized miRFP (miRFP) are set upstream of a nanoluciferase (nLuc) reporter. Constructs are expressed from an inducible Tet07 promoter. B: (Top) Representative assay data of relative light units (RLU) of each construct over time normalized to OD600. (Bottom) Schleif plot and associated trendlines of the top graph. C: Calculated elongation rate measurements of optYFP (n=9) and miRFP (n=4) ORFs. Error bars indicate SEM.

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  Fig. 2.. CGA-derived acute stalls negatively impact gene expression and increase elongation time in a dose-dependent manner. A: Diagram of optimal and CGA-containing constructs. Between 2 and 6 CGAs are inserted between the optYFP and nLuc ORFs. B: Protein expression of CGA constructs at T=60 min normalized to optimized control (2xCGA n=10, 3xCGA n=8, 4xCGA n=10, 5xCGA n=5, 6xCGA n=10). C: mRNA levels of CGA constructs at T=60 min normalized to optimized control. (n=3). C: Elongation delay of CGA-containing constructs compared to optimized control. (n=3). All error bars indicate SEM. All statistical significances were calculated for each construct using two-tailed paired Student’s t-Test against optYFP control.

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  Fig. 3.. HEL2 deletion rescues protein expression, mRNA levels, and elongation time. A: Protein expression fold change of CGA constructs in a hel2Δ vs WT background (2xCGA n=2, 4xCGA n=7, 6xCGA n=7). B: mRNA level fold change of CGA constructs in a hel2Δ vs WT background (n = 3). C: Elongation delay of CGA constructs in a hel2Δ vs WT background (n = 3). All error bars indicate SEM. All statistical significances were calculated for each construct using two-tailed paired Student’s t-Test against WT control.

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  Fig. 4.. Distributed stalls in the YFP ORF decrease protein expression, mRNA levels, and delays elongation time. A: Schematic of position of 21 leucine codons in YFP-nLuc construct, with numbers from 5’ end to 3’ end labeled below. The black box emphasizes the positions of No.7 and No.8 Leu codons B: Elongation delay of distributed stall constructs compared to optYFP (YFP[CTA] n=6, YFP[CTC] n=6, YFP[CTG] n=5, YFP[CTT] n=7). The first 20 out of 21 total optimal TTG leucine codons in optYFP are synonymously substituted to a nonoptimal codon specified in brackets. C: Protein expression of distributed stall constructs normalized to optYFP control (n=4 for all). D: mRNA levels of distributed stall constructs normalized to optYFP control (n=3 for all). E: (Left) Elongation delay measurements of chimeric constructs normalized to optYFP control. (n=4 for YFP[CTT], YFP[7,8CTT] and YFP[8CTT]; n=6 for YFP[1–7CTT]; n=9 for YFP[1–10CTT] and YFP[11–20CTT]). (Right) Elongation delay measurements of miRFP-YFP[CTT] normalized to miRFP-optYFP control (n=6). F: (Left) Protein expression chimeric constructs normalized to optYFP control. (n= 3 for YFP[CTT], YFP[7,8CTT] and YFP[8CTT]; n=6 for YFP[1–7CTT], n=8…

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  Fig. 5.. DHH1 deletion, but not HEL2 deletion, affects gene expression in substitution constructs. A: Protein expression of distributed stall constructs in dhh1Δ or hel2Δ background vs WT (n=5, 4, 5, 6, 7, and 3 from left to right). B: mRNA level fold change of distributed stall constructs in a dhh1Δ vs WT background (n = 5, 5, and 7 from left to right). C: Elongation delay of distributed stall constructs in dhh1Δ or hel2Δ background vs WT (n= 19, 4, 18, 6, 9, and 5 from left to right). All error bars indicate SEM. All statistical significances were calculated for each construct using two-tailed paired Student’s t-Test against WT control.