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. 2023 Feb 2;51(5):2377–2396. doi: 10.1093/nar/gkad040

Figure 5.

Figure 5.

Assessment of translational anomalies of arginine codon 2 and 5′-UTR position −1 in Librandom. (A) Effect of different synonymous codons in the second triplet of the CDS on rTR and predicted mRNA folding energy (efeC). Black circles within violins are mean rTR/efeC values. *** denote P-values < 10−16 in a Welch two sample t-test. (B) Relationship between relative triplet frequency in E. coli and rTR for the four synonymous triplets in arginine codon 2. (C) Effect of different bases in 5′-UTR position −1 on rTR and efeC. Black circles within violins are mean rTR/efeC values. *** denote P-values < 10−16 in a Welch two sample t-test. (D) Plasmids for the overexpression of native initiator tRNAfMet and mutants thereof (Supplementary Figure S6, Methods). Position 37 (3′-adjacent to the CAU anticodon) of tRNAfMet is mutated from A to C, G or T/U. (E) Growth of E. coli strains carrying plasmids for tRNAfMet overexpression in shake flask cultivations (LB, 37°C). Bars are mean doubling times of independent biological triplicate cultivations with standard deviation as error bars. Dashed lines are the mean doubling time of the respective strain without tRNA overexpression (i.e. empty vector control) with standard deviation as grey shaded areas. For tRNAfMet-A37C, doubling times were not determined (n.d.) due to severe growth inhibition (see main text). (F) Approximately 50 000 variants of Librandom were tested in the presence of overexpressed tRNAfMet variants in E. coli strains containing (WT) and lacking (ΔmetZWV) the chromosomal metZWV locus. (G) Impact of tRNAfMet mutations on the rTR of variants from Librandom. Displayed effects are log2-transformed fold-changes (log2 FC) of the average rTR of variants with a given base at 5′-UTR position −1 over the average rTR of variants with any other base at this position. Black arrows indicate complementarity between 5′-UTR position −1 and position 37 of the tRNAfMet variant. (H) Impact of complementarity between 5′-UTR position −1 and tRNAfMet position 37. Circles are log2-transformed fold-changes (log2 FC) of the average rTR of variants with complementarity or non-complementarity between mRNA and tRNA over the mean rTR of all variants in the same group (i.e. same tRNAfMet variant and strain). Bars are the mean log2 FCs of the three tRNAfMet variants for each case and strain with standard deviation as error bars.