Control of translation efficiency in yeast by codon-anticodon interactions

Abstract

The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNA(Arg(UCG)), and is associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.

FIGURE 1.

Systematic screen of the effects of synonymous codon repeats on firefly luciferase expression. (A) Schematic of reporter constructs used in these studies: Codon repeats (X) were inserted upstream of a firefly luciferase reporter gene either at position 4 of the coding region (X₄F), at position 314 in the Renilla-firefly fusion construct (RX₃₁₄F) or at position 4 of the coding region of Renilla luciferase (X₄R) or superfolder GFP (X₄G) (Pedelacq et al. 2006). X₄F and RX₃₁₄F were used for a systematic screen of the effects of 59 codons on expression. (B) Inhibitory effects of several codon repeats, but not CGA repeats, in X₄F are likely due to formation of strong RNA secondary structures. Luciferase activity for each 10-mer codon repeat at the N-terminus (%C) is plotted as a function of the free energy of the predicted RNA secondary structure from 1 to 50. Free energy of folding (nucleotides 1–50 of X₄F-reporter constructs containing 10 repeats of a particular codon for 59 of the 61 sense codons) (kcal/mol) was calculated using RNAstructure (Mathews et al. 1999) version 4.6 (http://rna.urmc.rochester.edu/RNAstructure.html). Similar results were obtained from calculations of free energy of folding from −4 to +37.

FIGURE 2.

CGA codon repeats inhibit expression in a dose-dependent manner. (A) Luciferase activity of three different reporter constructs (X₄F, RX₃₁₄F, and X₄R), containing (Arg)₁₀ insertions specified by each of the six Arg codons. (B) The relationship between CGA codon dose and luciferase activity was examined with (Arg)₁₀ inserts containing increasing CGA content, as illustrated in the schematic. (C) Firefly luciferase activity as a function of CGA codon dose in X₄F (Δ) and RX₃₁₄F (▪)—reporter constructs. (D) Firefly luciferase activity of X₄F-reporter constructs containing either no insertion, or insertion of four Arg residues, specified with the indicated CGA or AGA codons. Values were normalized to the X₄F-(AGA)₄ reporter. (E) Expression of integrated GFP constructs with three Arg residues inserted at amino acid 4 specified by AGA or CGA codons. GFP expression was assessed based on the distribution of yeast cells exhibiting different intensities of fluorescence emission at 530 nm.

FIGURE 3.

Suppression of CGA-mediated expression defects by its cognate tRNA. (A) The Arg codons are compared with respect to use in the genome, gene copy number of the decoding tRNA^Arg species and the requirement for wobble decoding. (B) Effect of overproduction of the indicated tRNA^Arg species from high copy plasmids on firefly luciferase activity of X₄F-reporter constructs containing (CGA)₁₀ insertions. Values were compared to activity of X₄F-(AGA)₁₀ luciferase in a strain with an empty vector. (C) Effect of overproduction of tRNA^Arg(ICG) from a high copy plasmid on firefly luciferase activity of RX₃₁₄F-reporter constructs containing (CGA)₁₀ insertions at amino acid 314. Values were normalized to (AGA)₁₀ overexpressing a vector control.

FIGURE 4.

Suppression of CGA-mediated expression defects by an exact base-pairing anticodon mutated tRNA^Arg(UCG). (A) Effect of indicated native and anticodon-mutated tRNA^Arg species, expressed on high copy plasmids, on firefly luciferase activity of X₄F-reporter constructs containing (CGA)₁₀ insertions. The parent tRNA of each anticodon-mutated tRNA^Arg is indicated by the first anticodon shown below the figure. Values were compared to activity of X₄F-(AGA)₁₀ luciferase in a strain with an empty vector. (B) Effect of an anticodon-mutated tRNA^Arg species, expressed on a high copy plasmid, on firefly luciferase activity of a RX₃₁₄F-reporter construct containing a (CGA)₁₀ insertion. (C) Suppression of the inhibitory effects of CGA codons by integrated copies of the mutant tRNA^Arg(UCG) species that decode CGA without wobble. Luciferase activity of X₄F-reporter constructs containing (Arg)₁₀ insertions, specified by (AGA)₁₀, [(CGA)₅(AGA)₅], or (CGA)₁₀, in yeast strains bearing an integrated copy of either of two native tRNA^Arg species, two nonnatural exact match tRNA^Arg species, or a vector control as indicated. Two individual integrant strains were analyzed in each case. Values were normalized to the average of the activity of X₄F-(AGA)₁₀ luciferase in the two integrant strains expressing a vector control. Error bars represent the standard deviation from three independent transformants, each assayed in triplicate. (D) A closer examination of expression of X₄F-reporter constructs containing (CGA)₁₀ insertions in indicated strains with an expanded scale of the y-axis. Values are normalized as in C.

FIGURE 5.

CGA-mediated inhibition affects firefly luciferase activity and protein levels, but does not result in reduced luciferase mRNA. Analysis of firefly luciferase protein, mRNA, and activity in X₄F-reporter constructs containing RGS-(His)₆-(Arg)₈ insertions, from yeast strains bearing the indicated constructs (lanes 1–10). Firefly luciferase protein was detected with antibody to RGS-(His)₆, with antibody to enolase serving as an internal control. In lanes 11–14, the top two panels contain dilutions of crude extracts expressing the X₄F- RGS-(His)₆ (AGA)₈ reporter construct (10, 7.5, 5.0, 2.5 μg). Firefly luciferase mRNA was measured by RT-PCR analysis using primers homologous to bases 21–46 and 148–173, as indicated on the diagram below the panel; sequences are given in Supplemental Table S3; measurements of Actin mRNA served as an internal control. In the third panel, lanes 11-14 contain RT-PCR reactions in which dilutions of DNase-treated RNA from X₄F-RGS-(His)₆ (CGA)₈-containing sample (0.6, 0.3, 0.15, 0.075 μg) were used in the RT reactions, with equal volumes of the RT reaction in each PCR reaction. Reverse transcriptase reactions in lanes 1–10 contain 0.3 μg total RNA input. Firefly luciferase mRNA amounts were first normalized to the levels of Actin mRNA and then quantified relative to the wild-type X₄F-reporter construct with no insertion; the FLuc mRNA levels in the wild-type strain were arbitrarily set to 100. The presence of RGS-(His)₆ is indicated by an asterisk (*). Firefly luciferase activity of X₄F-reporter constructs containing RGS-(His)₆-(Arg)₈ insertions was normalized to the wild-type X₄F-reporter construct with no insertion.

FIGURE 6.

The effects of CGA codons at amino acids 4 and 314 on full-length mRNA, examined by Northern analysis. (A) Diagram of the X₄R Renilla luciferase reporter indicating the position of the oligonucleotide probe used in panels B–D. Probe c is located at base pairs 682–705 in the 935 base-pair Renilla luciferase coding sequence. (B) Analysis of the effects of insertion of CGA codons at amino acid 4 upstream of Renilla luciferase on Renilla luciferase mRNA and activity. Renilla luciferase mRNA from X₄R reporter constructs containing RGS-(His)₆-(Arg)₈ was examined by Northern blots with the probe c indicated in panel A; U2 snRNA served as an internal control. Renilla luciferase mRNA levels were first normalized to levels of U2 snRNA and then compared to the wild-type Renilla luciferase mRNA containing RGS-(His)₆. In the lower panel, luciferase activity of Renilla luciferase reporter constructs containing RGS-(His)₆-(Arg)₈ insertions are shown. (Off, expression under repressing conditions.) Lanes 1–4 contain different amounts of input total RNA from the X₄R- RGS-(His)₆ (CGA)₈ bearing strain (20, 10, 5, 2.5 μg), while lanes 5–12 contain RNA (20 μg) from yeast strains bearing the indicated constructs. (C) Decoding of CGA codons with an anticodon-mutated tRNA^Arg species suppresses the CGA-mediated increase in Renilla luciferase mRNA. Northern blot analysis of Renilla luciferase mRNA from Renilla luciferase reporter constructs containing RGS-(His)₆-(Arg)₈, using Actin as an internal control. Strains expressed either a nonnatural exact match tRNA or a vector control. Luciferase mRNA amounts were normalized to the levels of Actin mRNA and then to RGS-(His)₆ (AGA)₈; In the titration indicated with the triangle, mRNA input from the strain X₄R- RGS-(His)₆ (CGA)₈ in a vector control was varied (20, 10, 5 μg). (D) Insertion of stop codons at amino acid 4 of Renilla luciferase resulted in a reduction in stable Renilla luciferase mRNA. Northern blot analysis of Renilla luciferase mRNA from indicated Renilla luciferase reporter constructs. Renilla luciferase mRNA was first normalized to levels of U1 snRNA and then to RLuc (wt) mRNA. (E) A diagram of the RX₃₁₄F reporter indicating the relative position of the probes used in panel F. Probe c is located at base pairs 682–705 in Renilla luciferase (935 base pairs), while probe d is located at base pairs 1564–1593 in the RX₃₁₄F reporter (2631 base pairs); this corresponds to base pairs 601–630 in firefly luciferase (1668 base pairs). Probe sequences are shown in Supplemental Table S3. (F) Analysis of the effects of insertion of CGA codons at amino acid 314 in the RX₃₁₄F reporter on Renilla-firefly luciferase mRNA. RNA from strains bearing RX₃₁₄F constructs with RGS-(His)_6, or RGS-(His)₆-(Arg)₈ encoded with either (AGA)₈, [(CGA)₄(AGA)₄], or (CGA)₈ as indicated was examined by Northern blots with the probes indicated in panel E. U2 snRNA served as an internal control.

FIGURE 7.

CGA codon pairs are more inhibitory than single CGA codons. (A,B) The effects of the arrangement of five CGA codons within an (Arg)₁₀ insert on Renilla luciferase activity were compared to each other and to Renilla luciferase activity from constructs containing (Arg)₁₀ insertions specified by (AGA)₁₀ or (CGA)₁₀. (A) The arrangements of Arg CGA codons with the number of CGA codons and codon pairs indicated and (B) the resulting luciferase activity with lanes corresponding to constructs in A. Reported values were corrected to an independently transcribed firefly luciferase gene, and then normalized to (AGA)₁₀. (C) Schematic of the arrangement of CGA codons in test constructs designed to examine the effects of single CGA codons, pairs of CGA codons, and alternating CGA codons. (D) Relative luciferase activity of firefly luciferase reporter constructs containing (Arg)₁₆ insertions with increasing CGA content arranged either as single codons or codon pairs. Reported values were corrected to an independently transcribed Renilla luciferase gene, and then normalized to (AGA)₁₆. (E) Relative luciferase activity of Renilla luciferase reporter constructs containing (Arg)₁₆ insertions with increasing CGA content arranged either as single codons or codon pairs. Reported values were corrected to an independently transcribed firefly luciferase gene, and then normalized to (AGA)₁₆. (F) Relative luciferase activity of Renilla luciferase reporter constructs containing (Arg)₁₆ insertions with increasing CGA content. CGA codons are clustered either as single codons separated by two AGA codons or as alternating codons (CGA–AGA). Reported values were corrected to an independently transcribed firefly luciferase gene, and then normalized to (AGA)₁₆.