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. 2017 May 2;114(18):4745-4750.
doi: 10.1073/pnas.1614896114. Epub 2017 Apr 17.

Case for the genetic code as a triplet of triplets

Fabienne F V Chevance  1 , Kelly T Hughes  2
Affiliations

Case for the genetic code as a triplet of triplets

Fabienne F V Chevance et al. Proc Natl Acad Sci U S A. .

Abstract

The efficiency of codon translation in vivo is controlled by many factors, including codon context. At a site early in the Salmonella flgM gene, the effects on translation of replacing codons Thr6 and Pro8 of flgM with synonymous alternates produced a 600-fold range in FlgM activity. Synonymous changes at Thr6 and Leu9 resulted in a twofold range in FlgM activity. The level of FlgM activity produced by any codon arrangement was directly proportional to the degree of in vivo ribosome stalling at synonymous codons. Synonymous codon suppressors that corrected the effect of a translation-defective synonymous flgM allele were restricted to two codons flanking the translation-defective codon. The various codon arrangements had no apparent effects on flgM mRNA stability or predicted mRNA secondary structures. Our data suggest that efficient mRNA translation is determined by a triplet-of-triplet genetic code. That is, the efficiency of translating a particular codon is influenced by the nature of the immediately adjacent flanking codons. A model explains these codon-context effects by suggesting that codon recognition by elongation factor-bound aminoacyl-tRNA is initiated by hydrogen bond interactions between the first two nucleotides of the codon and anticodon and then is stabilized by base-stacking energy over three successive codons.

Keywords: context effects on translation; genetic code; synonymous codon effects; tRNA; translation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
The steps of mRNA translation from codon recognition at the A (Acceptor) site by GTP-bound elongation factor EF-Tu complexed with aminoacyl-tRNA to the peptidyl transfer step. (Adapted from ref. .)
Fig. 1.
Fig. 1.
A model showing the effect of Watson–Crick base pairing and the influence of base stacking with bases in the anticodon loop and adjacent bases in the mRNA sequence on codon–anticodon stabilization in the accommodation step in translation and showing how translation can be affected by codons adjacent to the codon being translated (context).
Fig. 2.
Fig. 2.
The effects of silent mutations on FlgM inhibitory activity of the σ28-dependent transcription of a PmotA–lux reporter construct. (A) FlgM inhibitory activity is presented as 1/luminescence levels measured for each strain (SI Materials and Methods) divided by the levels measured for the wild-type flgM strains with ACC at position 6 and CCU at position 8. The boxes depicting inhibitory levels are shaded red for increased FlgM activity and blue for decreased activity to highlight the severe translation defect of the Thr6 ACC–Pro8 CCG context. (B) FlgM inhibitory activity was determined for isogenic strains that contain the six synonymous Leu9 positions placed in context with the severe Pro8 CCG substitution (SI Materials and Methods) and compared with the wild type. (C) FlgM inhibitory activity was determined for 24 isogenic strains that contain each of four threonine codons (ACN) at amino acid position 6 in combination with each of six leucine codons (UUG/A and CUN) at amino acid position 9 of flgM and was compared with the wild-type activity.
Fig. S2.
Fig. S2.
The effects of synonymous codon changes on mRNA levels. (A) Quantitative real-time PCR for Thr6-Pro8 synonymous strain constructs. Effects of specific synonymous changes (bars are numbered from the left): Thr6 ACU/Pro8 CCU (bar 1), Thr6 ACC/Pro8 CCU (bar 2), Thr6 ACG/Pro8 CCU (bar 3), Thr6 ACU/Pro8 CCG (bar 4), Thr6 ACC/Pro8 CCG (bar 5), and Thr6 ACG/Pro8 CCG (bar 6) on flgM (Top) and fliC (Middle) reporter mRNA levels. (Bottom) Quantitative RT-PCR on hisG control mRNA. Error bars represent the SD of the means. One-way ANOVA followed by Tukey test showed which datasets were statistically different. ns, not significant; *P = 0.05; **P = 0.01; ***P = 0.001. Only fliC mRNA levels were significantly different at 95% confidence interval levels. Both flgM and hisG mRNA levels were not significantly different for all constructs tested. (B) Quantitative real-time PCR for Thr6-Leu9 synonymous strain constructs. Effects of specific synonymous changes: Thr6 ACC/Leu9 UUG (bar 1), Thr6 ACG/Leu9 UUG (bar 2), Thr6 ACC/Leu9 CUC (bar 3), Thr6 ACG/Leu9 CUC (bar 4), Thr6 ACC/Leu9 CUG (bar 5), and Thr6 ACG/Leu9 CUG (bar 6) on flgM (Top) and fliC reporter (Middle) mRNA levels. (Bottom) Quantitative RT-PCR on hisG control mRNA. Errors bars represent the SD of the means. One-way ANOVA followed by Tukey test showed which datasets were statistically different. ns, not significant; *P = 0.05; **P = 0.01; ***P = 0.001. Only fliC mRNA levels were significantly different at 95% confidence interval levels. flgM and hisG mRNA levels were not significantly different for all constructs tested.
Fig. 3.
Fig. 3.
The effects of codon context on flgM and flgM–lacZ translation. (A) A Western blot using anti-LacZ antibody shows the effect of reduced flgM translation in a flgM–lacZ construct with the CCG codon at position 8 of flgM compared with the wild-type CCU codon at position 8. Lane 1 shows LacZ levels in a wild-type strain with a flgM–lacZ operon fusion (flgM5222::MudJ) where flgM and lacZ are translated separately. Lanes 2 through 6 show levels of LacZ proteins in strains carrying a flgM–lacZ gene fusion (flgM7512::MudK). Lanes 2 and 3 show the full-length FlgM–LacZ fusion using wild-type flgM coding sequences (Pro8 = CCU). The strains used in lanes 3 and 5 carry a σ28 H14D substitution that results in increased PflgM transcription and facilitates visualization of the differences in expression of the lower LacZ band (24). The effect of the CCU-to-CCG change at Pro8 on FlgM–LacZ expression is shown in lanes 4 and 5. The strain used for lane 6 carries a UAG stop codon at the Pro8 position in flgM. (B) Effects of mutant SerT tRNA on FlgM–lacZ expression. Lane 1 is the same as in lane 1 in A. Lanes 2 and 3 are the FlgM–lacZ translational fusion with Pro8 CCU and Pro8 CCG, respectively. Lanes 4 through 7 are samples from strains that carry the σ28 H14D substitution resulting in increased PflgM transcription that are used only in this gel to optimize the visualization of the LacZ protein band (24). Lanes 4 and 5 are Pro8 CCU and Pro8 CCG, respectively, in a serT mutant background. Lanes 6 and 7 are Pro8 CCU and Pro8 CCG, respectively, in a serT+ background.
Fig. 4.
Fig. 4.
Suppression of the flgM Pro8 CCG allele by doped oligonucleotide synonymous codon mutagenesis of codons 2–25. In a strain unable to assemble the flagellar motor (a hook basal body-mutant strain, HBB−), FlgM is not secreted, resulting in the inhibition of σ28-dependent PfliC–lac expression and a Lac− phenotype on MacConkey lactose indicator plates. Introduction of the flgM Pro8 CCG results in reduced FlgM levels, increased σ28-dependent PfliC–lac expression, and a Lac+ phenotype on MacConkey lactose indicator plates. Doped oligonucleotide mutagenesis that introduced synonymous changes in amino acids 2–25 (excluding Pro8) was performed on the flgM Pro8 CCG, HBB− mutant strain. More than 10,000 recombinant strains were screened for increased FlgM activity that resulted in a Lac− phenotype. Mutants resulting in a Lac+ phenotype are shown in red and occurred at amino acid codons Thr6, Ser7, and Leu9. Mutants with a slight Lac+ phenotype are in blue and occurred at codons Ser2 and Val12. Ten independent colonies with the parental Lac− phenotype were sequenced and are shown in black. The numbers in parenthesis are the number of independent isolates that were sequenced.
Fig. 5.
Fig. 5.
Position effects of the Pro8 CCU-to-CCG change on the expression and mRNA stability of flgM(codons 1–85)–lacZ and fliA(codons 1–60)–flgM(codons 61–85)–lacZ gene fusions. (A) Fusions of codons 1–85 of flgM to lacZ were made without and with duplicate insertions of codons Thr6-Ser7-Pro8-Leu9-Lys10 after amino acid codons 26 and 56. (B) Fusions of codons 1–60 of fliA fused to codons 61–85 of flgM fused to lacZ were made with insertions of flgM codons Thr6-Ser7-Pro8-Leu9-Lys10 after amino acid codons 5, 26, and 56. The effects of the synonymous Pro8 CCU-to-CCG change on the expression and mRNA stability for all six constructs were determined. The fusions are deleted for the C-terminal region of flgM that contains an RBS, and no ATG or GTG start codon precedes the lacZ-coding segment of each fusion. Expression of the different fusion constructs was determined by β-Gal assay under arabinose inducing conditions and mRNA levels were determined as described in SI Materials and Methods.
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