Significance The genetic code for life is a triplet base code. It is known that adjacent codons can influence translation of a given codon and that codon pair biases occur throughout nature. We show that mRNA translation at a given codon can be affected by the two previous codons. Data presented here support a model in which the evolutionary selection pressure on a single codon is over five successive codons, including synonymous codons. This work provides a foundation for the interpretation of how single DNA base changes might affect translation over multiple codons and should be considered in the characterization of the effects of DNA base changes on human disease.

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.

The DNA sequence is transcribed to form mRNA, which then is translated into protein by ribosomes. The genetic code consists of 64 triplet RNA codons that specify the 20 amino acids and sites of translation termination (stop codons). The decoding process involves interaction between a specific codon in the mRNA and the three-base anticodon of the cognate aminoacyl-tRNA (bases 36, 35, and 34 within the anticodon loop) (1). The code is redundant, in that many amino acids are specified by two or more triplet sequences. In addition, codons specifying a particular amino acid are recognized by single or multiple tRNAs. Most tRNA species recognize codons that differ in the third or “wobble” position. Modifications of tRNA bases also contribute to tRNA recognition of single or multiple anticodons as the tRNA enters the ribosome at the A site (1). Codons are read in the 5′–3′ direction of the mRNA by sequential interactions with tRNA aligned 3′–5′ vis-à-vis each codon. Stacking-related wobble codon recognition is mediated by extensive modification of position 34 of the tRNA codon.

Other base modifications stabilize the interaction of the tRNA with ribosomal A site, i.e., methylation of a G at position 37 prevents tRNA slippage, which would result in a shift in the translation reading frame (1). Fig. S1 illustrates the initial translation steps starting with codon recognition and proceeding to peptidyl transfer. GTP-bound elongation factor Tu (EF-Tu) brings aminoacyl-tRNA to the A site of the ribosome. Watson–Crick pairing at the first two bases of the codon initiates EF-Tu–tRNA docking followed by triplet stabilization at the third (wobble) position. GTP hydrolysis by EF-Tu is a slow step in the process providing a test of the correctness of triplet recognition. If an incorrect match is sensed, the tRNA is rejected, and the process begins again. A correct match leads to the acceptance of the incoming tRNA followed by peptidyl transfer. Correct codon–anticodon recognition results from stabilization of the EF-Tu aminoacyl-tRNA at the A site, which is determined by the Watson–Crick pairing, plus energy of stacking between bases of the mRNA and tRNA.

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. 2.)

Based on the work presented here, we propose a model to explain the effects of codon context on mRNA translation efficiency. In the model, bases of the anticodon loop of the tRNA, which are often heavily modified, and mRNA bases flanking the codon contribute to the stacking energy of codon–anticodon recognition (shaded gray in Fig. 1). These interactions combine to determine the efficiency at which a given codon is translated.

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).

The tRNA modifications vary throughout the three kingdoms of life (3) and could affect codon–anticodon pairing. The differences in tRNA modifications could account for differences in synonymous codon biases and for the effects of codon context (the ability to translate specific triplet bases relative to specific neighboring codons) on translation among different species. Here, using in vivo genetic systems of Salmonella, we demonstrate that the translation of a specific codon depends on the nature of the codons flanking both the 5′ and 3′ sides of the translated codon, thus generating higher-order genetic codes for proteins that can include codon pairs and codon triplets.

The effect of the flanking codons on the translation of a specific codon varies from insignificant to profound. It has been known for decades that highly expressed genes use highly biased codon pairs, which can vary from one species to the next. The speed of translation depends heavily on flanking codons (4).

Results The Effects of Synonymous Codon Changes Adjacent to Ser7 on FlgM Activity. The assay system described below involves the FlgM protein of Salmonella, which coordinates flagellar gene expression and flagellum assembly. FlgM binds and inhibits a flagellum-specific transcription factor, σ28, before completion of the flagellar motor (the hook basal body). When the flagellar motor is completed, unassociated FlgM is conducted out of the cell through the completed flagellar motor, thereby freeing internal σ28 and stimulating the transcription of flagellar genes whose products are needed following motor completion. These proteins include the external flagellar filament and chemosensory proteins (5, 6). Our work on the effect of synonymous mutations in codon translation began with the isolation of a tRNA mutant in which translation of flgM mRNA was reduced. The mutant resulted from a G–A base change at position 10 within the D-stem of the essential serT tRNA [tRNA(cmo5UGA)(Ser)] (7). This mutation resulted in a defect in the translation of the UCA codon for amino acid 7 of FlgM (8). There are dozens of UCA codons in the more than 50 genes required for flagellum production and chemotaxis. Therefore we were surprised that this serT mutant was perfectly motile. Why did the mutant apparently translate the many UCA codons of all the flagellar genes normally but fail to translate the UCA codon efficiently in flgM? It seemed possible that the defect in flgM translation might be caused by context effects of codons flanking the UCA codon for amino acid 7 (Ser7) of FlgM. The Ser7 UCA codon in flgM is flanked at the 5′ side by a threonine codon, ACC (Thr6), and at the 3′ side by the proline CCU (Pro8) codon. We wanted to determine whether codons adjacent to the UCA codon for Ser7 affected its translation. To avoid effects of amino acid substitutions on FlgM activity, we focused on synonymous codon changes. Synonymous changes at Thr6 and Pro8 would not affect the amino acid sequence of FlgM protein. Any effects of adjacent, synonymous substitutions would be limited to effects on mRNA secondary structure and mRNA stability or translation. If synonymous changes at Thr6 and Pro8 affected the ability of the ribosome to translate the Ser7 UCA triplet because of codon-context effects, then the levels of FlgM protein could increase or decrease. Slight changes in the level of this protein are detected easily because of the effect of FlgM on the regulation of flagellar gene expression. Four triplets encode Thr (ACN), and four others encode Pro (CCN). Thus, there are 16 possible silent change combinations at codons 6 and 8 of flgM that do not change the translated FlgM amino acid sequence. We assayed the effects of these 16 combinations on FlgM production in a wild-type serT+ background by scoring σ28-dependent transcription of luminescence operon transcribed from a σ28-dependent promoter (P motA -lux) (Fig. 2). Reduction of FlgM causes an increase in σ28-dependent transcription. Fig. 2. The effects of silent mutations on FlgM inhibitory activity of the σ28-dependent transcription of a P motA –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. The effects of synonymous changes at Thr6 and Pro8 on FlgM activity are presented in Fig. 2A. The 16 context backgrounds exhibited a >600-fold range in FlgM activity. We conclude that codon context has a profound effect on flgM mRNA translation through the UCA Ser7 codon. Remarkably, various pairs of synonymous changes at positions Thr6 ACN and Pro8 CCN showed synergistic context effects on translation of the central Ser7 UCA codon. The single change of the Pro8 codon in the wild-type sequence from CCU to CCG (with the Thr6 codon unchanged) resulted in about a 20-fold reduction in FlgM activity (FlgM activity was 0.053 of wild type). The single change in the wild-type sequence of the Thr6 codon from ACC to ACU resulted in an 11-fold increase in FlgM activity. If the synonymous changes at Thr6 ACN and Pro8 CCN affected flgM translation independently, then the effect of combining codon changes at these positions should be additive. However, the combination of CCU to CCG at Pro8 and ACC to ACU at Thr6 produced a synergistic effect on flgM mRNA translation that resulted in a 34-fold increase in FlgM activity. The synonymous codon changes had no apparent effects on flgM mRNA secondary structure or stability (SI Results, Table S1, Dataset S1, and Fig. S2). Table S1. Spearman correlation between predicted folding energy and expression, in overlapping windows each of length 30 nt, numbered relative to start codon 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. Taken together, our results demonstrate that translation of the Ser7 UCA codon is dependent on both the 5′ and 3′ flanking codons and that the synergistic effects on translation of the central codon depend on context effects extending over three codons. Because these synonymous changes had no apparent effect on flgM mRNA secondary structure or stability, our results suggests that the ribosome senses at least three consecutive triplet codons during the process of translation. The Reduced Translation at Pro8 CCG of flgM Is Context Dependent. The severely reduced FlgM activity caused by the synergistic effects of the Pro8 CCG codon and the Thr6 ACC codon were not seen with the other Thr6 synonymous codons. When the Thr6 ACC codon was replaced by ACU, ACA, or ACG in combination with CCG at Pro8, we observed FlgM activities increased by 34-, 11-, and 6.7-fold above wild type, respectively (Fig. 2A). If the defect in translation of flgM mRNA with the Pro8 CCG substitution was caused by context, we wondered if translation of Pro8 CCG could be sensitive to synonymous changes on the 3′ side of Pro8 at Leu9. If synonymous changes at Leu9 did not affect Pro8 CCG translation, then FlgM activities would be expected to remain low. All six Leu9 codons were constructed adjacent to Pro8 CCG with wild-type codons at Thr6 (ACC) and Ser7 (UCA), and the cells were assessed for FlgM activity. An 11-fold range in FlgM activity levels was observed for the six Leu9 codons when adjacent to Pro8 CCG (Thr6 ACC), suggesting that translation at CCG could be context dependent on both 5′ and 3′ codons (Fig. 2B). The Severe Translation Defect at Thr6 ACC-Ser7 UCA-Pro8 CCG of FlgM Results in Ribosome Pausing in Vivo. In the tests described above, it was assumed that the 20-fold reduction in the in vivo FlgM activity for the CCU-to-CCG synonymous codon change at Pro8 was caused by reduced translation of flgM mRNA. To test this assumption, we set up an assay for ribosome pausing to see if the reduction in flgM translation correlated with a pause in ribosome translation of Pro8 CCG in vivo. The pausing assay uses a flgM–lacZ gene fusion that allows lacZ to be translated independently of flgM if translation is stalled at Pro8. The flgM and lacZ genes were placed in a single operon where they were transcribed into a single mRNA but could be translated independently. Cells with this structure were phenotypically FlgM+, LacZ+, and the LacZ protein was easily identified by Western blot using anti-LacZ antibody, as shown in Fig. 3A, lane 1. The flgM and lacZ genes were fused so that the 5′ end of the flgM coding sequence contained a ribosome-binding site (RBS), and the lacZ part of the fusion started with a GTG start codon. With no stalling, translation would produce a single fusion protein including both FlgM and LacZ sequences. If ribosomes stalled upstream in flgM at Pro8 CCG codon, this stalling would expose the downstream RBS and allow lacZ to be translated independently of flgM. When full-length flgM was fused to lacZ, the larger FlgM–LacZ protein fusion product was observed (Fig. 3A, lane 2). Increased transcription of the flgM promoter allowed us to detect a small amount of LacZ in addition to the FlgM–LacZ fusion (Fig. 3A, lane 3). The lower-molecular-weight (MW) LacZ band could result from translation restart at lacZ or from protein degradation. Introduction of the CCU–CCG codon change at Pro8 resulted in decreased FlgM–LacZ fusion product (Fig. 3A, lane 4). However, in a strain allowing increased transcription from the flgM promoter, the CCU–CCG codon change at Pro8 resulted in a substantial increase of the lower band corresponding to LacZ alone as well as in the FlgM–LacZ fusion band (Fig. 3A, lane 5). The presence of the lower-MW LacZ band resulting from the CCU–CCG synonymous change at Pro8 is consistent with a pause in translation at Pro8 that resulted in translation reinitiation downstream within the flgM–lacZ coding sequence to produce the shorter LacZ band observed. To confirm this observation, a UAG stop codon was introduced at the Pro8 position in the flgM–lacZ gene fusion construct. As expected, the Pro8 CCU-to-UAG (stop codon) change produced no full-length FlgM–LacZ protein; only the lower-MW LacZ band was observed, indicating translation of lacZ independent of flgM translation in the flgM–lacZ fusion construct (Fig. 3A, lane 6). This result also indicated that the flgM–lacZ mRNA was stable in the absence of full flgM translation. These data are consistent with the stalling of translation at codon Pro8 CCG, which resulted in a substantial reinitiation of translation downstream and producing the lower-MW product. 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 P flgM 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 P flgM 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. We also tested the effect of the serT tRNA mutant that previously was shown to be defective in translating the Ser7 UCA codon of flgM on flgM–lacZ translation of the Pro8 CCU and Pro8 CCG constructs (Fig. 3B). The control of a flgM–lacZ transcriptional fusion in which flgM and lacZ are translated independently (Fig. 3A, lane 1) is repeated in Fig. 3B, lane 1. As shown in Fig. 3B, reduction in the level of FlgM–LacZ fusion was observed by the Pro8 CCU–CCG change (Fig. 3B, lanes 2 and 3). In a strain background with increased transcription from the flgM promoter, the lower-MW LacZ band accumulates in the presence of Pro8 CCG change (Fig. 3B, lanes 4 and 5). In the serT mutant we observe a decrease in FlgM–LacZ levels but not in the lower-MW LacZ band (Fig. 3B, lanes 6 and 7). These results demonstrate that the serT mutant is significantly defective in flgM–lacZ translation relative to translation of lacZ. The Effects of Synonymous Codon Changes at Thr6 and Leu9 on FlgM Activity. We looked for synergistic effects of synonymous changes at codons Thr6 and Leu9, which are separated by two triplet codons. In this case, there are four synonymous codons for Thr (ACN) and six codons for Leu (UUA/G and UCN), including 24 possible silent change combinations that do not change the translated FlgM amino acid sequence. We assayed these 24 combinations for FlgM regulatory activity; the results are shown in Fig. 2C. Here the range in FlgM regulatory activity varied from a 10% reduction in FlgM activity to a 22-fold increase in FlgM activity over wild type. For the most part, the contributions of synonymous changes at Thr6 and Leu9 were additive. We did not observe any dramatic synergistic effects as we did with synonymous combinations of Thr6 and Pro8 codons on Ser7 reading. However, we did observe small synergistic effects resulting in an approximately twofold synergistic effect on FlgM activity for the Thr6 ACG substitution in combination with the Leu9 CUU, CUC, CUA, and CUG substitutions (Fig. 2C). The remaining changes in FlgM activity caused by the synonymous Thr6 and Leu9 combinations could be accounted for by additive changes of the single synonymous substitutions at Thr6 and Leu9. This result supports the conclusion that if the ribosome does sense more than three consecutive triplet codons during the process of translation, the effect is small. Isolation of Synonymous Suppressor Mutations of Pro8 CCG. The above data suggest that the translation of a specific codon can be influenced significantly by the adjacent two codons. Neither mRNA secondary structure nor mRNA stability appeared to be a factor. However, it is very difficult to rule completely out the effects of mRNA secondary structure and mRNA stability on translation. In an attempt to rule out further the effects of mRNA secondary structure and stability on translation, we set up a genetic assay to look for more long-distance effects of flgM mRNA changes affecting codons for amino acids 2 through 25 on translation of the Pro8 CCG allele of flgM. A doped oligonucleotide mutagenesis of the region of flgM mRNA encoding amino acids 2–25 was performed. Technical details of the doped oligonucleotide synthesis limited our experiment to codons 2–25. The oligonucleotide mutagenesis was designed so that, on average, each oligo had one synonymous codon change for amino acids 2–25. We reasoned that if the 20-fold reduced FlgM activity of the Pro8 CCU-to-CCG change was caused by an effect on mRNA secondary structure, suppressors might be obtained throughout the amino acid 2–25 region that significantly increased FlgM activity. Alternatively, if translation of a given codon is influenced by the two adjacent codons, we expected significant suppression of the Pro8 CCG translation defect to occur in the region including the two codons before (Thr6 and Ser7) and the two codons after (Leu9 and Lys10) Pro8. The results of the doped synonymous mutagenesis of amino acids 2–25 in the Pro8 CCG background are presented in Fig. 4. Synonymous changes that significantly suppressed the FlgM inhibitory activity of Pro8 CCG are shown in red. Changes resulting in partial suppression are shown in blue. To show that the doped oligonucleotide mutagenesis covered the entire region, 10 colonies that showed no suppression of the Pro8 CCG allele were sequenced and are shown in black. 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 P fliC –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 P fliC –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. The synonymous changes that suppressed the Pro8 CCG alleles to give wild-type FlgM activity included all three possible synonymous substitutions at Thr6, all three possible synonymous substitutions at Ser7, and the UUG-to-UUA substitution at Leu9, which we earlier showed increased FlgM activity of the Pro8 CCG allele sixfold. For technical reasons, the CUN Leu9 synonymous substitutions, which also suppressed the CCG translation defect (Fig. 2B), were not included in this experiment. Only synonymous changes in the two codons (Thr6 and Ser7) preceding Pro8 and the Leu9 codon following Pro8 CCG resulted in restoration to levels near that of wild-type FlgM inhibitory activity. These results support our assertion that the efficiency of translation of a specific codon is dependent on the flanking two codons and that our observed effects are caused primarily by codon context and not by mRNA secondary structure. The Effect of Gene Location on the CCU-to-CCG Translation Defect in the Context of Thr-Ser-Pro-Leu-Lys. Codon bias effects on translation are enriched at the N terminus of genes (9⇓⇓⇓–13). We wondered if the translating ribosome might transition from a translation initiation mode through the N terminus, which is strongly influenced by the presence of rare codons, codon context, mRNA secondary structure, tRNA modifications, and other translation factors, into a more robust translation machine that is less affected by these factors. Recently, Boël et al. determined that the codon choice in the initial 20 codons of Escherichia coli transcripts correlated with translation efficiency and mRNA stability (14). To test whether the translational defect of Pro8 CCG in flgM was caused by its N-terminal location and whether translational reinitiation affected mRNA stability, we constructed two fusion constructs with the Thr6-Ser7-Pro8-Leu9-Lys10 codons of flgM at different gene locations with either CCU or CCG at the Pro position. One construct placed codons 1–85 of flgM to a lacZ under control of an arabinose-inducible promoter to remove any possible autoregulatory effects of flgM on its own transcription and lacked both the RBS region at the C terminus of flgM and a separate start codon for lacZ (Fig. 5). The second construct was identical except that codons 1–60 of flgM in the fusion were replaced with codons 1–60 of fliA. By measuring β-gal activity under arabinose-inducing conditions, we are measuring a direct effect of the Pro8 CCG change on flgM–lacZ translation. 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. For the flgM(1–85)–lacZ construct, a change from Pro8 CCU to CCG reduced β-gal activity about sevenfold, but in this case mRNA levels also were reduced by about fourfold (Fig. 5A). Insertion of a duplicated copy of codons Thr6 through Lys10 of flgM after amino acid codon 26 still produced a translation defect of 30% when the middle Pro codon was changed from CCU to CCG in the inserted fragment. The insertion of flgM codons Thr6 through Lys10 alone resulted in a twofold reduction in mRNA levels, but here the CCU to CCG change had no effect on mRNA levels. Inserting flgM codons Thr6 through Lys10 after codon 56 had no effect on either β-gal activity or mRNA levels. For the fliA(1–60)–flgM(61–85)–lacZ construct, flgM codons Thr6 through Lys10 were separately inserted after amino acids 5, 26, and 56 with either the CCU or CCG proline. Both β-gal and mRNA levels were reduced similarly with Pro-CCG at all three positions. These results support our hypothesis that translation of a given codon can be influenced by the two preceding codons and that a given codon can influence the translation of the following two codons, at least within the first 60 codons of a gene.

SI Results Effect of Synonymous Changes at Codons Thr6 and Pro8 of flgM on mRNA Levels. To rule out effects of the Thr6 and Pro8 substitutions on flgM mRNA stability, we determined the flgM mRNA levels for the different Thr6–Pro8 synonymous combinations by quantitative RT-PCR (Fig. S2). The substitution combinations with a wide range of FlgM activities chosen for comparison with the wild type (Thr6:ACC Pro8:CCU) were Thr6:ACU Pro8:CCU (FlgM activity increased 11-fold), Thr6:ACG Pro8:CCU (FlgM activity increased 13-fold), Thr6:ACU Pro8:CCG (FlgM activity decreased 20-fold), Thr6:ACC Pro8:CCG (FlgM activity increased 34-fold), and Thr6:ACG Pro8:CCG (FlgM activity increased 6.7-fold). No significant effect on flgM mRNA levels was observed, suggesting that the synonymous substitutions did not affect flgM mRNA stability. We also assayed mRNA levels of the σ28-dependent fliC gene. As expected, the levels of fliC mRNA varied in accordance with the FlgM activities reported in Fig. 2A. Effect of Synonymous Changes at Codons Thr6 and Leu9 of flgM on mRNA Levels. We also tested the effects of Thr6–Leu9 synonymous combinations on flgM mRNA stability for six combinations, including wild-type. The substitution combinations with a range of FlgM activities chosen for comparison with the wild type (Thr6:ACC Leu9:UUG) were Thr6:ACG Leu9:UUG (FlgM activity increased 13-fold), Thr6:ACC Leu9:CUC (FlgM activity increased 1.1-fold), Thr6:ACG Leu9:CUC (FlgM activity increased 4.5-fold), Thr6:ACC Leu9:CUG (FlgM activity decreased 0.9-fold), and Thr6:ACG Leu9:CUG FlgM activity (increased 4.9-fold). A slight (∼20%) reduction for only the Thr6:ACC Leu9:CUC was observed. No other synonymous codon effects on flgM mRNA levels were observed, suggesting that the synonymous substitutions did not affect flgM mRNA stability (Fig. S2). Again the mRNA levels for the σ28-dependent fliC gene were determined for the Thr6 Leu9 synonymous combinations, and the levels of fliC mRNA varied in accordance with FlgM activities reported in Fig. 2C. Predicted mRNA Secondary Structural Effects. To test for possible mRNA secondary structure effects, predicted folding energies of the flgM mRNA were calculated on a window-by-window basis for all 16 codon variants. Predicted folding energies were calculated using RNAfold of ViennaRNA package v2.0.2 (25) on overlapping 30-nt windows. To check whether the codon variants of flgM cluster in the RNA secondary space, an alignment- and folding-free clustering algorithm for RNA secondary structures, called “NoFold,” was used (26). The algorithm clustered the 16 variants into four clusters. Details of each cluster and the consensus structure for each cluster are presented in Dataset S1. We find no clear pattern between the identities of the sequences within a cluster and the luminescence patterns observed in Fig. 1A. We analyzed the predicted secondary structure of the 16 variants for Thr6 and Pro8 of flgM mRNA to determine if the rank order of folding energies in any window correlates with the rank order of expression in the experimental data. Although the predicted mRNA folding energies in windows around the Ser6 codon varied considerably with synonymous changes in codon context, we found no clear pattern between the folding energies of flgM mRNA and its activity based on luminescence assays (Table S1 and Dataset S1). This result suggests that variations in mRNA secondary structure are not the cause for the variations in FlgM activity observed.

Discussion We describe a sensitive translation assay involving a regulatory gene, flgM, and show that translation of a particular codon is heavily influenced by two adjacent codons 5′ and one adjacent codon 3′ to the codon being translated. The expression assays used in this study took advantage of FlgM being a regulatory gene that works in a one:one stoichiometry with the σ28 transcription factor. Thus, small changes in FlgM protein levels would result in signal amplification on σ28-dependent transcription. This amplification of signal allowed us to observe the effects of small changes in flgM mRNA translation on the expression of reporter constructs. We analyzed synonymous codon-context effects on translation and conclude that the translation of a given codon can be influenced significantly by up to two flanking codons. That is, when EF-Tu bound to an aminoacyl-tRNA enters the A site at a particular codon, two preceding codons can affect the correct-fit determination by EF-Tu and the resulting translation efficiency of that codon. In a similar fashion, that codon can influence the translation of the next two codons being translated. Since 1980 investigation of the effects of codon context on translation has focused primarily on codon pairs. In their seminal paper, Bossi and Roth determined that the translation of a UAG codon by an amber suppresser tRNA was highly influenced by the adjacent codons (15). It has been known for decades that highly expressed genes in bacteria and yeast exhibit codon pair biases. Modern high-throughput technologies (genetic selection being the classical high-throughput technology) can identify adjacent codon pairs through tens of thousands of combinations that modulate translation efficiency (16). Changing codon-pair biases on a genomic scale has proven an effective way to attenuate viruses in making effective live vaccines (17). We assume that the effects of biased codon pairs are caused by changes in the translation speed of various proteins (4). Similar methods that include codon pair speeds in designing attenuated essential genes may aid in the design of live bacterial vaccines. Our data suggest that in thinking about context effects one should consider codon triplets in addition to codon pairs. Our data demonstrate that the translation of a specific codon can depend on the two codons flanking the codon being translated in a nonadditive manner. After the second codon of a given gene, both the P-site and E-site of the ribosome are occupied when elongation factor EF-Tu bound to a charged aminoacyl-tRNA species brings nascent charged tRNA to an empty A site. We propose that upon docking an aminoacyl-tRNA to the A site, the efficiency with which EF-Tu translates that codon is influenced by adjacent codons and bound tRNA species in addition to the specific codon–anticodon interaction at the A site. Our working model for the effect of two adjacent codons on specific codon translation is diagrammed in Fig. 1. In our model, GTP-bound elongation factor EF-Tu (purple) directs a charged tRNA to the A site of the ribosome through Watson–Crick pairing of the first two bases in the translating triplet. The efficiency with which this codon is translated depends on the stability of the anticodon–codon interactions. We propose that this stability is dependent on base-stacking energy, which is influenced by at least the two flanking codons in the mRNA. We also propose that the stability of the anticodon–codon interactions is influenced by modifications of the tRNAs that affect interaction with the ribosomal A, P, and E sites and by protein or noncoding RNA translation factors. Synonymous changes will lead to differences in translation rates that, especially when different tRNAs are used, have different binding efficiencies, abundances, and charging rates and result in differential mRNA stabilities. In addition, the same tRNA reads different codons with different efficiencies, as was determined in an in vivo translational speedometer assay system (4). We conclude that codon recognition is initiated by codon–anticodon hydrogen bonding between the first and second bases of the translated codon followed by sensing the correct fit at the wobble base and base-stacking interactions contributed by the preceding two codons and bound tRNAs. The tRNA molecules of every organism are modified extensively, and the majority of modifications occur at the antiwobble position of the anticodon loop and at the base immediately 3′ to the anticodon (18). [Thirteen other bases positions are modified to a lesser extent in tRNA species of E. coli and Salmonella enterica (7).] The base adjacent to the 3′ anticodon position, the “cardinal nucleotide,” also varies among species and is thought to affect codon recognition significantly (19). These modifications influence the stacking energy of the bases during codon–anticodon pairing (3). The translation proofreading steps catalyzed by EF-Tu and EF-G, which “sense” hydrogen bonding and stacking energy to determine if the correct codon–anticodon pairing has occurred, are influenced by the adjacent codons, possibly resulting in the codon-context effects we observe. Moreover, many tRNA-modifying proteins are present in only one of the three kingdoms of life (1). Thus, specific tRNA modifications that affect wobble base recognition and contribute to the base-stacking forces during translation can determine specific codon-context effects by adjacent synonymous codons on specific codon translation. Such effects of specific tRNA modifications on codon translation could account for the different codon pair biases observed in species that are evolutionarily distant (possessing different specific tRNA modifications) and also could account for the difficulty in expressing proteins in heterologous systems, i.e., expressing proteins from plant and mammalian systems in bacteria. The MiaA (i6A37) modification has recently been shown to affect mRNA translation in E. coli in a codon context-dependent manner, supporting our overall hypothesis (20). The translation of proline codons in the mgtL peptide transcript of Salmonella was recently shown to be affected by mutations defective in ribosomal proteins L27, L31, elongation factor EF-P, and TrmD, which catalyzes the m1G37 methylation of proline tRNA (21). Modification of tRNA species in E. coli also has been shown to vary with the growth phase of the cell (22). Specific codon-context effects could represent translation domains of life based on tRNA modifications. The difficulty for natural selection would be in finding codon optimization for a given gene. If the speed through a codon is dependent on the 5′ and 3′ flanking codons, and the flanking codons are dependent on their 5′ and 3′ flanking codons, then selection pressure on a single codon is exerted over five successive codons, which represent 615 or 844,596,301 codon combinations. If modified tRNAs interact with bases in a codon context-dependent manner that differs among species depending on differences in tRNA modifications, ribosome sequences, and ribosomal and translation factor proteins, it is easy to understand why many genes are poorly expressed in heterologous expression systems in which codon use is the primary factor in the design of coding sequences for foreign protein expression. The potential impact of differences in tRNA modifications represents a significant challenge in designing genes for maximal expression whether by natural selection or in the laboratory.

Materials and Methods Strains and oligonucleotides used in this work are listed in SI Materials and Methods. Strain constructions and quantitative real-time PCR were performed as described in SI Materials and Methods. Expression of luciferase was measured as luminescence levels on a POLARstar OPTIMA luminometer from BMG Labtech. β-Gal activities were determined as described previously (23).

SI Materials and Methods Bacterial Strains, Plasmids, and Media. All strains are derived from Salmonella Typhimurium strain LT2 and are listed below in the section Strains and Oligonucleotides. Cells were cultured in lysis broth (LB) (per liter: 10 g Bacto tryptone, 5 g Bacto yeast extract, and 5 g NaCl). Antibiotics were added to LB at the following final concentrations: 100 µg/mL sodium ampicillin, 12.5 µg/mL chloramphenicol, 15 µg/mL tetracycline-HCl, or 0.5 μg/mL anhydrotetracycline (ATc). l-Arabinose was supplemented to 0.2% (wt/vol) as needed. Amresco agar (12 g/L) was added for solid media. The generalized transducing phage of Salmonella typhimurium P22 HT105/1 int-201 was used in all transductional crosses (27). ATc plates used to select for tetracycline-sensitive recombinants were prepared as follows: Flask A consisted of 12 g Amresco agar, 5 g Bacto tryptone, and 5 g yeast extract in 500 mL H 2 O; flask B consisted of 10 g NaCl, 10 g NaH 2 PO 4 ⋅H 2 O in 500 mL H 2 O. Following autoclave sterilization, flasks A and B were cooled to ca. 55 °C. To flask B was added 2.5 mL of 0.2 mg/mL ATc in 50% ethanol (stored at 4 °C), 5 mL of 2.4 mg/mL fusaric acid in dimethylformamide (stored at −20 °C), and 5 mL 20 mM ZnCl 2 . Flasks A and B were mixed before pouring. ATc was protected from light and stored at 4 °C before use. Strain Constructions. Targeted chromosomal mutagenesis was carried out via the tetRA insertion and replacement with the λ-Red recombinase system as described previously (28). Primers were synthesized by Integrated DNA Technologies, the DNA synthesis Core Facility of the University of Utah, or Eton Biosciences, Inc. All PCR and fillin reactions were performed using a proofreading polymerase (Phusion; Thermo Fisher Scientific). Recombinant products mediated by λ-Red were confirmed by sequencing analyses at the DNA Sequencing Core Facility of the University of Utah or Eton Biosciences, Inc. Construction of FlgM Thr6 and Pro8 Synonymous Codon Combinations. Synonymous changes at amino acid positions Thr6 and Pro8 in FlgM were constructed by λ-Red–mediated recombination using a large primer, FlgM-ACN-TCA-CCN, which was made double stranded with primer flgMfillinNNN using Phusion polymerase. The DNA fragment was ethanol precipitated, resuspended in 10 μL of Milli-Q water (Millipore), and electroporated into strain TH18446, which contained a tetRA cassette deleting amino acids 6–8 of FlgM [pKD46/fla-5398 (serT) ΔflgM8081::tetRA (ΔAA6–8) ΔflgG-L2157 fliC5050::MudJ]. The cells were plated on ATc plates at 37 °C. Recombinants were isolated on LB plates and were assessed on MacConkey lactose and X-gal plates for color variation; the colonies with the largest range of colors were sent for sequencing analysis to obtain the maximum codon combination in the initial screen. Codon combinations not obtained in the initial screen were constructed individually to cover the 16 codon combinations with the same sequence primer as for FlgM-ACN-TCA-CCN but with the specific codons instead of N. Strain combinations in a serT+ background were obtained by moving the serT+ allele from strain LT2 into the 16 strains produced by P22 transduction as described above and selection for growth at 42 °C. Construction of FlgM Pro8::CCG-Leu9 and FlgM Thr6-Leu9 Synonymous Codon Combinations. Construction of FlgM Pro8::CCG Leu9 synonymous codons was done as described above but using primers FlgM leu9TTA and FlgM leu9CTN filled in with primer flgMfillinNNN. Synonymous changes at Thre6 and Leu9 of FlgM were obtained using the strategy described above, with primers FlgMThre6-SP-Leu9 and FlgMThre6-SPLeu9TTA filled in with primer flgMfillinNNN. The DNA fragments were ethanol precipitated, resuspended in 10 μL Milli-Q water, electroporated into strain TH20628 [pKD46/ΔflgM8081::tetRA (deleted for amino acids 6–8) ΔflgG-L2157 fliC5050::MudJ fljBenx vh2], and plated on ATc plates at 37 °C. Recombinants were isolated and assessed on X-gal and MacConkey lactose indicator plates for color variations, and a range of colored colony variants were sent for sequencing analysis to maximize different codon combinations by DNA sequence analysis. Remaining strains not isolated were constructed individually to cover the 24 codon combinations with the same sequence primers as for FlgMThre6-SP-Leu9 and FlgMThre6-SPLeu9TTA but with the specific codon instead of N. Synonymous codons Thre6 (ACN) combined with Leu9 (TTG) in FlgM had been obtained previously, as described above. Construction of FlgM–β-Gal Fusions and Introduction of a Stop Codon at Amino Acid 8 of FlgM–lacZ. A LacZ translational fusion was engineered at the end of FlgM in FlgM Thr6 and Pro8 synonymous codon combinations by adding a MudK fusion (29) to the end of the flgM coding sequence, with a GTG codon at the beginning of lacZ (flgM-MudK) to allow for restart of lacZ translation independent of flgM translation. A stop codon was introduced at amino acid 8 (AA8) of flgM-MudK as a control to check for the restart of lacZ translation independent of flgM translation. The stop codon was introduced by λ-Red using primers FlgMAA8stopTGA filled in with primer flgMfillin NNN and strain pKD46/TH18916 [flgM6683::tetRA (inserted between amino acids 5 and 6) flgM7512::MudK (flgM-MudK)] as the recipient. FlgM AA8::TGA-MudK was transduced into a ΔflgG-L2157 background. Isolation of Suppressors of the Pro8-CCG Translation-Defective Allele. Strain TH21455 [flgM::tetRA (deleted for amino acids 2–25) ΔflgG-L2157 fljBenx vh2] was constructed by targeted chromosomal mutagenesis using a DNA fragment generated by PCR amplification of a Tn10 template and primers FlgMstarttetR and FlgMAA26tetA. Plasmid pkD46 was introduced by electroporation selecting ampicillin at 30 °C, yielding strain TH21456. The oligonucleotide FlgM1, which covers the N-terminal region of FlgM and carries the Pro8-CCG translation-defective allele, was doped at the Core Facility of the University of Utah as follows: t ggc cgc tac aac gta acc ctc gat gag gat aaa taa atg agc(T)att(M) gac(T) cgt(C) acc(D) tca(B) ccg ttg(A) aaa(G) ccc(D) gtt(V) agc(T) act(V) gtc(D) cag(A) acg(H) cgc(D) gaa(G) acc(D) agc(T) gac(T) acg(H) ccg(H) gta(B) caa aaa acg cgt cag gaa aaa acg tcc gcc gcg acg agc g. The third base was doped with 97, 98, or 99% of the correct base (third base indicated) and was replaced with the mixture in parenthesis. Single-base doping was 99% correct and was 1% A, C, G, or T. Two-base doping was for only a single triplet “att” at 98% with mixture M (A,C) added at 2%. Three-base doping is 97% correct base with mixtures H (A,C,T), B (C,G,T), V (A,C,G), or D (A,G,T) added at 3%. This oligonucleotide was made double-stranded by primer fillin with the primer flgM1fillin and was electroporated into strain TH21456 and plated on ATc plates at 42 °C. Twelve hundred colonies were picked and patched on ATc plates, replica-printed onto Tz-Lac indicator plates, screened for Tz-Lac red colonies, and analyzed by sequencing. Construction of P ara -flgM(1–85)-lacZ Fusions Without and With a Thr-Ser-Pro-Leu-Lys Codon Segment Inserted After Codon 26 or 56 of the flgM Coding Sequence. The isolation of a flgM(1–89)::MudK insertion that results in the fusion of codons 1–85 of flgM to lacZ has been described (30). The tetRA element was inserted after codons 26 and 56 of the flgM(1–89)::MudK construct and was replaced with the Thr6-Ser7-Pro8-Leu9-Lys10 codon segment of flgM with either Pro8 CCU or CCG as described. Amino acid codons 6–10 of flgM were inserted after codons for AA26 and AA56 of flgM using primers flgMcodon 26, flgMcodon26-ccg, flgMcodon56, and flgMcodon56CCG. The flgM(1–85)::MudK constructs were PCR amplified and placed under the control of the P araBAD promoter by λ-Red recombination with a P araBAD -tetRA-MudK insertion at the araBAD locus by replacement of the tetRA element with the flgM sequence as described (31). The strains were deleted for the flhDC operon to remove any effects of flagellar gene expression. Targeted mutagenesis DNA fragment was amplified from strains TH21437 (flgMCCU-AA85-mudK) and TH21438 (flgM CCC-AA8-mudK) using the primers ARAFLGM and MuRK-2 and were electroporated into strain TH21576 [pKD46 (ApR)/ΔaraBAD1068::tetRA-MudK ΔflhDC7902::FRT], yielding strains TH21606 and TH21608, respectively. Amino acid codons 6–10 of flgM were inserted after codons AA26 and AA56 of flgM using the primers flgMcodon 26, flgMcodon26-ccg, flgMcodon56, and flgMcodon56CCG. Construction of P ara -fliA (1–60)-flgM(61–85)-lacZ Fusions Without and With a Thr-Ser-Pro-Leu-Lys Codon Segment Inserted After Codon 5, 26, or 56 of the fliA Coding Sequence. A tetRA element was first inserted at the beginning of flgM in strain TH21606 [ΔflhDC7902::FRT ΔaraBAD2013::flgM (codons 1–85)-MudK] by λ-Red recombination using primers BADTetR and flgM21tetA. The first 60 codons of FliA then were introduced just before codon 61 of flgM-MudK by λ-Red recombination using recipient strain TH22716 and the primers araBfliA GTG and FliA60-flgM61rev, yielding strain TH22719. TetRA elements were inserted after codons 5, 26, and 56 of fliA in recipient strain TH22719 using the primer pairs ParafliA5tetR and fliA5tetA, fliA26tetR and fliA26tetA, and fliA56tetR and fliA56flgMtetA to yield strains TH22721, TH22722, and TH22723, respectively. The tetRA elements were replaced by codons 6–0 of flgM (with Pro8CCU or Pro8CCG) after codons 5, 26, or 56 of fliA using the primers ParafliA5-M6-10wt or ParafliA5-M6-10ccg filled with fliA5fill, primers fliA26-M6-10wt or fliA26-M6-10ccg filled in with fliAfill26, or fliA56-M6-10wt or fliA56-M6-10ccg filled with fliA56fill, yielding strains TH22733, TH22734, TH22735, TH22736, TH22737, and TH22738. FlgM and σ28 Activity Measurement Assays. The pRG19 (P motAB -luxCDEF-CmR) plasmid carrying a class 3 flagellar promoter fused to the luciferase operon (32) was first introduced into all strains to be assayed. Strains were grown overnight in triplicate in LB medium with 12.5 µg/mL chloramphenicol at 30 °C. Overnight cultures (2 μL) were inoculated into 198 μL of LB + Cm in 96-well plates. Each sample was grown in quadruplicate on the 96-well plate. Replicates samples were conducted on different plates. For each plate, a reference sample was used, and fluorescence values were compared with that reference. Cultures were grown in the 96-well plates on a platform shaker at 30 °C for 3 h to an OD of 0.4. Luminescence and OD were measured using a POLARstar OPTIMA (BMG LabTech) at 30 °C. Luminescence was expressed per OD and relative to the control sample. The σ28 activity is the activity of class 3 luciferase expression per OD value. FlgM activity is the inverse of the class 3 luciferase per OD value. β-Gal Assay. Overnight cultures were diluted 100-fold into fresh LB medium. Cultures then were incubated with shaking at 37 °C until the contents reached a midlog-phase density of OD 0.6. Cultures were put on ice, spun down, and resuspended in 3 mL of cold, buffered saline. Culture samples of 0.1 mL were added to 0.4 mL of buffered saline and 0.55 mL of complete Z-buffer (Z-buffer plus 5 μL of 10% SDS and 100 μL of CHCl 3 ) (23). The assay was continued as described previously (23). All assays were performed three to six times using independent cultures. Activity is expressed in nanomoles per minute for OD 650 . Real-Time PCR Assays. RNA isolation was performed for three independent biological replicates using the RNeasy minikit (Qiagen). For removal of genomic DNA, RNA was treated with DNase I for 30 min at 37 °C using the DNA-free RNA kit (Zymo Research). Subsequently, RNA samples were reverse transcribed according to the RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using the EvaGreen quantitative real-time PCR master mix (Bio-Rad) and the primers FlgMfwdRTpcr and FlgMrevRTpcr for flgM; RT-PCR fliCfwd and RT-PCR fliCrev for fliC; RT-PCR lacZfwd and RT-PCR lacZrev for the construction lacZ fusions; hisG-multiRT-fwd and hisG-multiRT-rev for hisG; rpoA-RT-fwd and rpoA-RT-rev for rpoA; and gyrB-RT-fwd and gyrB-RT-rev for gyrB. Experiments were performed on a CFX96 real-time PCR instrument (Bio-Rad). Relative changes in mRNA levels were analyzed according to the Pfaffl method (33) and were normalized against the transcript levels of the reference genes hisG, rpoA, and gyrB. Western Blots Against lacZ. SDS/PAGE (7.5%) was performed using standard procedures on a Bio-Rad system. Immunoblotting was conducted using anti−β-gal purified monoclonal antibody (Promega) followed by secondary IRDye800 goat anti-mouse (LI-COR). Antigen–antibody complexes were visualized by infrared detection using the LI-COR Odyssey imaging system. Strains and Oligonucleotides Used. Strains. CF313 = flgM8326(codons 6–8 = ACT-TCA-CCT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF314 = flgM + (codons 6–8 = ACC-TCA-CCT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF315 = flgM8327(codons 6–8 = ACA-TCA-CCT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF316 = flgM8328(codons 6–8 = ACG-TCA-CCT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF317 = flgM8329(codons 6–8 = ACT-TCA-CCC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF318 = flgM8330(codons 6–8 = ACC-TCA-CCC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF319 = flgM8331(codons 6–8 = ACA-TCA-CCC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF320 = flgM8332(codons 6–8 = ACG-TCA-CCC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF321 = flgM8333(codons 6–8 = ACT-TCA-CCA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF322 = flgM8334(codons 6–8 = ACC-TCA-CCA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF323 = flgM8335(codons 6–8 = ACA-TCA-CCA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF324 = flgM8336(codons 6–8 = ACG-TCA-CCA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF325 = flgM8337(codons 6–8 = ACT-TCA-CCG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF326 = flgM8252(codons 6–8 = ACC-TCA-CCG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF327 = flgM8338(codons 6–8 = ACA-TCA-CCG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF328 = flgM8339(codons 6–8 = ACG-TCA-CCG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF346 = flgM7512::MudK ΔflgG-L2157 fliA*5226

CF358 = flgM8252(codon8 = CCG) flgM7512::MudK ΔflgG-L2157 fliA*5226

CF403 = flgM7512::MudK serT

CF414 = flgM8252(codon 8 = CCG) flgM7512::MudK serT

CF417 = flgM7512::MudK

CF418 = flgM8252(codon 8 = CCG) flgM7512::MudK

CF504 = flgM8340(codons 8,9 = CCG,TTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF505 = flgM8341(codons 8,9 = CCG,CTT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF507 = flgM8343(codons 8,9 = CCG,CTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF509 = flgM8345(codons 8,9 = CCG,CTG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF511 = flgM8347(codons 8,9 = CCG,CTC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF555 = flgM8348(codons 6,9 = ACT,CTT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF556 = flgM8349(AA6,9 = ACC,CTT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF557 = flgM8350(codons 6,9 = ACA,CTT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF558 = flgM8351(codons 6,9 = ACG,CTT) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF559 = flgM8352(codons 6,9 = ACT,CTC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF560 = flgM8353(codons 6,9 = ACC,CTC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF561 = flgM8354(codons 6,9 = ACA,CTC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF562 = flgM8355(codons 6,9 = ACG,CTC) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF563 = flgM8356(codons 6,9 = ACT,CTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF564 = flgM8357(codons 6,9 = ACC,CTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF565 = flgM8358(codons 6,9 = ACA,CTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF566 = flgM8359(codons 6,9 = ACG,CTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF567 = flgM8360(codons 6,9 = ACT,CTG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF568 = flgM8361(codons 6,9 = ACC,CTG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF569 = flgM8361(codons 6,9 = ACA,CTG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF570 = flgM8363(codons 6,9 = ACG,CTG) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF571 = flgM8364(codons 6,9 = ACT,TTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF572 = flgM8365(codons 6,9 = ACC,TTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF573 = flgM8366(codons 6,9 = ACA,TTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

CF574 = flgM8367(codons 6,9 = ACG,TTA) ΔflgG-L2157 fliC5050::MudJ fljB e,n,x vh2

TH2779 = flgM5222::MudJ

TH19989 = flgM7512::MudK flgM8157(codon 8 = TGA) ΔflgG-L2157 fliA*5226

TH21437 = flgM5208::MudK ΔflgG-L2157 fljB e,n,x vh2

TH21575 = ΔaraBAD1068::tetRA-MudK ΔflhDC7902::FRT

TH21576 = pKD46 (Ap R )/ΔaraBAD1068::tetRA-MudK ΔflhDC7902::FRT

TH21606 = ΔflhDC7902::FRT ΔaraBAD2013::flgM (codons 1–85 of flgM Pro8 = CCU)-MudK

TH21608 = ΔflhDC7902::FRT ΔaraBAD2014::flgM (codons 1–85 of flgM Pro8 = CCG)-MudK

TH22590 = ΔflhDC7902::FRT ΔaraBAD2030::flgM (codons 1–85 of flgM with insertion of flgM Thr6-Ser7-Pro8CCU-Leu9-Lys10 coding segment after codon 26 of flgM)-MudK

TH22591 = ΔflhDC7902::FRT ΔaraBAD2031::flgM (codons 1–85 of flgM with insertion of flgM Thr6-Ser7-Pro8CCG-Leu9-Lys10 coding segment after codon 56 of flgM)-MudK

TH22592 = ΔflhDC7902::FRT ΔaraBAD2032::flgM (codons 1–85 of flgM with insertion of flgM Thr6-Ser7-Pro8CCU-Leu9-Lys10 coding segment after codon 56 of flgM)-MudK

TH22593 = ΔflhDC7902::FRT ΔaraBAD2033::flgM (codons 1–85 of flgM with insertion of flgM Thr6-Ser7-Pro8CCG-Leu9-Lys10 coding segment after codon 56 of flgM)-MudK

TH22716 = pKD46 (Ap R )/ΔflhDC7902::FRT ΔaraBAD2043::tetRA- flgM (codons 21–85)-MudK

TH22717 = ΔflhDC7902::FRT ΔaraBAD2044::fliA(codons 1–60)- flgM (codons 61–85)-MudK

TH22719 = pKD46 (Ap R )/ΔflhDC7902::FRT ΔaraBAD2044::fliA(codons 1–60)- flgM (codons 61–85)-MudK

TH22721= ΔflhDC7902::FRT ΔaraBAD2046::fliA(codons 1–5)-tetRA-fliA(codons 6–60)-flgM (codons 61–85)-MudK

TH22722= ΔflhDC7902::FRT ΔaraBAD2047::fliA(codons 1–26)-tetRA-fliA(codons 27–60)-flgM (codons 61–85)-MudK

TH22723= ΔflhDC7902::FRT ΔaraBAD2048::fliA(codons 1–56)-tetRA-fliA(codons 57–60)-flgM (codons 61–85)-MudK

TH22727= pKD46 (Ap R )/ΔflhDC7902::FRT ΔaraBAD2046::fliA(codons 1–5)-tetRA-fliA(codons 6–60)-flgM (codons 61–85)-MudK

TH22728= pKD46 (Ap R )/ΔflhDC7902::FRT ΔaraBAD2047::fliA(codons 1–26)-tetRA-fliA(codons 27–60)-flgM (codons 61–85)-MudK

TH22729= pKD46 (Ap R )/ΔflhDC7902::FRT ΔaraBAD2048::fliA(codons 1–56)-tetRA-fliA(codons 57–60)-flgM (codons 61–85)-MudK

TH22733= ΔflhDC7902::FRT ΔaraBAD2052::fliA(codons 1–5)-flgM (codons Thr6-Ser7-Pro8CCU-Leu9-Lys10)-fliA(codons 6–60)-flgM (codons 61–85)-MudK

TH22734= ΔflhDC7902::FRT ΔaraBAD2053::fliA(codons 1–5)-flgM (codons Thr6-Ser7-Pro8CCG-Leu9-Lys10)-fliA(codons 6–60)-flgM (codons 61–85)-MudK

TH22735= ΔflhDC7902::FRT ΔaraBAD2054::fliA(codons 1–26)-flgM (codons Thr6-Ser7-Pro8CCU-Leu9-Lys10)-fliA(codons 27–60)-flgM (codons 61–85)-MudK

TH22736= ΔflhDC7902::FRT ΔaraBAD2055::fliA(codons 1–26)-flgM (codons Thr6-Ser7-Pro8CCG-Leu9-Lys10)-fliA(codons 27–60)-flgM (codons 61–85)-MudK

TH22737= ΔflhDC7902::FRT ΔaraBAD2056::fliA(codons 1–56)-flgM (codons Thr6-Ser7-Pro8CCU-Leu9-Lys10)-fliA(codons 57–60)-flgM (codons 61–85)-MudK

TH22738= ΔflhDC7902::FRT ΔaraBAD2057::fliA(codons 1–56)-flgM (codons Thr6-Ser7-Pro8CCG-Leu9-Lys10)-fliA(codons 57–60)-flgM (codons 61–85)-MudK. Oligonucleotides. FlgM-ACN-TCA-CCN: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt ACN TCA CCN ttga aac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

flgMfillinNNN: 5′-t cgc tgg ttt cgc gcg tct g-3′

FlgM leu9TTA: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt acc tca ccg tt a a aac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

FlgM leu9CTN: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt acc tca ccg ctn aaac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

FlgMThre6-SP-Leu9: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt ACN TCA CCT CTN aaac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

FlgMThre6-SPLeu9TTA: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt ACN TCA CCT TTA aaac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

FlgMAA8stopTGA: 5′-acg taa ccc tcg atg agg ata aat aaa tga gca ttg acc gt acc tca TGA t tga aac ccg tta gca ctg tcc aga cgc gcg aaa cca gcg a-3′

FlgMstarttetR: 5′-t ggc cgc tac aac gta acc ctc gat gag gat aaa taa atg TT AAG ACC CAC TTT CAC ATT-3′

FlgMAA26tetA: 5′-ggc gct cgt cgc ggc gga cgt ttt ttc ctg acg cgt ttt ttg CTA AGC ACT TGT CTC CTG-3′

ARAFLGM: 5′-actgtttctccatacctgtttttctggatggagtaagacgatgagcattgaccgtacctc-3′

MuRK-2: 5′-gattaagttgggtaacgccag-3′

flgMcodon 26: 5′-t agc act gtc cag acg cgc gaa acc agc gac acg ccg gta acc tca cct ttg aaa caa aaa acg cgt cag gaa aaa acg tcc gcc gcg acg agcg-3′

flgMcodon26-ccg: 5′-t agc act gtc cag acg cgc gaa acc agc gac acg ccg gta acc tca ccG ttg aaa caa aaa acg cgt cag gaa aaa acg tcc gcc gcg acg agcg-3′

flgMtetR56: 5′-g tta agc gac gcg caa gcg aag ctc atg cag cca ggc gtc TTA AGA CCC ACT TTC ACA TT-3′

flgMtetA56: 5′-agccgtttttaatgcttcgacgcgttccatatt aat gtc cgt CTA AGC ACT TGT CTC CTG-3′

flgMcodon56: 5′-g tta agc gac gcg caa gcg aag ctc atg cag cca ggc gtc acc tca cct ttg aaa agc gac att aat atg gaa cgc gtc gaa gca tta aaa acg g-3′

flgMcodon56CCG: 5′-g tta agc gac gcg caa gcg aag ctc atg cag cca ggc gtc acc tca ccG ttg aaa agc gac att aat atg gaa cgc gtc gaa gca tta aaa acg g-3′

BADTetR: 5′-ACTGTTTCTCCATACCTGTTTTTCTGGATGGAGTAAGACGTTAAGACCCACTTTCACATT-3′

flgM21tetA: 5′-GGCGGACGTTTTTTCCTGACGCGTTTTTTGTACCGGCGTGTCCTAAGCACTTGTCTCCTG-3′

araBfliA GTG: 5′-ACTGTTtCTcCataCctGTtttTCTgGaTggAGTAAGaCGgtgaattcactgtataccgc-3′

FliA60-flgM61rev: 5′-ctcaccgttacggatagccgtttttaatgcttcgacgcgttc GTC ATA TCG GTC GAC CGC A-3′

ParafliA5tetR: 5′-ctgtttttctggatggagtaagacg GTG AAT TCA CTG TAT TTAAGACCCACTTTCACATT-3′

fliA5tetA: 5′-acgctgccacagcgagtgtttatccattacaccttcagcggt CTAAGCACTTGTCTCCTG-3′

fliA26tetR: 5′-acactcgctgtggcagcgttatgtaccgctggtgcgtcacTTAAGACCCACTTTCACATT-3′

fliA26tetA: 5′-ttccacgctcgccggcaatcgcacctgcaggcgcaatgcttcCTAAGCACTTGTCTCCTG-3′

fliA56tetR: 5′-tctgctacaagcgggc ggc atc ggg tta tta aat gcg gtc TTA AGA CCC ACT TTC ACA TT-3′

fliA56flgMtetA: 5′-gat agc cgt ttt taa tgc ttc gac gcg ttc gtc ata tcg gtc CTAAGCACTTGTCTCCTG-3′

ParafliA5-M6-10wt: 5′-ctgtttttctggatggagtaagacg GTG AAT TCA CTG TAT acc tca cct ttg aaa acc gct gaa ggt gta atg gat aaa cac tcg ctg tgg cag c-3′

ParafliA5-M6-10ccg: 5′-ctgtttttctggatggagtaagacg GTG AAT TCA CTG TAT acc tca ccG ttg aaa acc gct gaa ggt gta atg gat aaa cac tcg ctg tgg cag c-3′

fliA5fill: 5′-gtacataacg ctg cca cag cga gtg ttt-3′

fliA26-M6-10wt: 5′-acactcgctgtggcagcgttatgtaccgctggtgcgtcac acc tca cct ttg aaa gaagcattgcgcctgcaggtgcgattgccggcgagcgtggaa-3′

fliA26-M6-10ccg: 5′-acactcgctgtggcagcgttatgtaccgctggtgcgtcac acc tca ccG ttg aaa gaagcattgcgcctgcaggtgcgattgccggcgagcgtggaa-3′

fliAfill26: 5′-gtccag ttc cacgctcgccggcaat-3′

fliA56-M6-10wt: 5′-tctgctacaagcgggc ggc atc ggg tta tta aat gcg gtc acc tca cct ttg aaa gaccgatatgacgaacgcgtcgaagcattaaaaacggcta-3′

fliA56-M6-10ccg: 5′-tctgctacaagcgggc ggc atc ggg tta tta aat gcg gtc acc tca ccG ttg aaa gaccgatatgacgaacgcgtcgaagcattaaaaacggcta-3′

fliA56fill: 5′-gga tag ccg ttt tta atg ctt cg-3′

flgMfwdRTpcr: 5′-aacggctatccgtaacggtgagtt-3′

FlgMrevRTpcr: 5′-ttactctgtaagtagctctgcgcc-3′

RT-PCR fliCfwd: 5′-gtcgctgttgacccagaataa-3′

RT-PCRfliCrev: 5′-cgtctttcgcgctgttgata-3′

RT-PCR lacZfwd: 5′-atc ttc ctg agg ccg ata ct-3′

RT-PCR lacZ rev: 5′-cgg att gac cgt aat ggg ata g-3′

hisG-multiRT-fw: 5′-gaa aac atg ccg att gat atc ctg-3′

hisG-multiRT-rv: 5′-agc acg tttt cgc cga taa tac-3′

rpoA-RT-fw: 5′-cgc cct gtt gac gat ctg g-3′

rpoA-RT-rv: 5′-ttt acc caa gtt agg cgt ctt aag-3′

gyrB-RT-fw: 5′-ctg ctc aaa gag ctg gtg tat ca-3′

gyrB-RT-rv: 5′-agc gcg tta cag tct gct cat-3′.

Acknowledgments We thank Premal Shah and Joshua Plotkin at the University of Pennsylvania for the mRNA secondary structural analysis; John Roth, Lionello Bossi, Nara Figueroa-Bossi, Winfried Boos, Urs Jenal, and Jon Seger for helpful discussions; Henri Grosjean for educating us on the role of base stacking and tRNA modifications in mRNA translation; and the University of Utah 2014 Microbial Genetics BIO5255 class for their help in producing the data presented in Fig. 4. This work was supported by start-up funds from the University of Utah and Public Health Service Grant GM056141 from the National Institutes of Health.