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Outwitting EF-Tu and the ribosome: translation with d-amino acids.

Achenbach J, Jahnz M, Bethge L, Paal K, Jung M, Schuster M, Albrecht R, Jarosch F, Nierhaus KH, Klussmann S - Nucleic Acids Res. (2015)

Bottom Line: We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases.The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation.Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.

View Article: PubMed Central - PubMed

Affiliation: NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany jachenbach@noxxon.com.

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The effect of tRNA properties on d-aa incorporation. Single incorporation assays with four different tRNAs misacylated with six different l- and d-amino acids were performed in independent duplicates (triplicates for Lys). Template ‘G1’ was used for translation with tRNAGlyu (high EF-Tu and A-site affinity) and tRNAGlytp (low EF-Tu affinity, high A-site affinity) and template ‘O’ for tRNATyru (low EF-Tu and A-site affinity) and tRNATyrtp (high EF-Tu affinity, low A-site affinity). The anticodon on both tRNATyr was changed to UCA to enable opal stop codon suppression. (A) Autoradiograms of the gels. Remarkable differences in the d-aa incorporation efficiencies depending on EF-Tu affinity become apparent. (B) Signal intensities normalized to the respective reaction with l-aa-tRNAGlyu (lane 2 in (A)) are plotted. Control signals were subtracted from d-aa reaction signals prior to normalization. For clarity, the y-axis scaling is compressed above 150%. Error bars show the range of determined data.
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Figure 4: The effect of tRNA properties on d-aa incorporation. Single incorporation assays with four different tRNAs misacylated with six different l- and d-amino acids were performed in independent duplicates (triplicates for Lys). Template ‘G1’ was used for translation with tRNAGlyu (high EF-Tu and A-site affinity) and tRNAGlytp (low EF-Tu affinity, high A-site affinity) and template ‘O’ for tRNATyru (low EF-Tu and A-site affinity) and tRNATyrtp (high EF-Tu affinity, low A-site affinity). The anticodon on both tRNATyr was changed to UCA to enable opal stop codon suppression. (A) Autoradiograms of the gels. Remarkable differences in the d-aa incorporation efficiencies depending on EF-Tu affinity become apparent. (B) Signal intensities normalized to the respective reaction with l-aa-tRNAGlyu (lane 2 in (A)) are plotted. Control signals were subtracted from d-aa reaction signals prior to normalization. For clarity, the y-axis scaling is compressed above 150%. Error bars show the range of determined data.

Mentions: Since a full-length product band may arise from unspecific readthrough of a hungry codon or due to small enantiomeric impurities found in the d-aa derivatives (l-aa content <0.1–0.96%), each reaction was accompanied by a control dedicated to show the maximum possible extent of unspecific full-length product formation. The control reactions contained the same absolute amounts of tRNAGlyu, precisely deacyl-tRNAGlyu and 1% of the respective l-aa-tRNAGlyu to simulate the worst-case enantiomeric impurity. For evaluation, we assessed the intensities of the full-length product bands and subtracted the control signals from the d-aa signals, which we then related to the l-aa signals (Figure 3). Despite the relatively high variance between replicates, the data clearly point into the same direction: d-amino acids can reproducibly be incorporated with a reasonable efficiency. The variance is related to the fact that each data point results from two independent aminoacylation reactions (charging of l- and d-amino acid) and three independent translation reactions (control, l-aa, d-aa), followed by purification and analysis. Replicates were in no case conducted in parallel.


Outwitting EF-Tu and the ribosome: translation with d-amino acids.

Achenbach J, Jahnz M, Bethge L, Paal K, Jung M, Schuster M, Albrecht R, Jarosch F, Nierhaus KH, Klussmann S - Nucleic Acids Res. (2015)

The effect of tRNA properties on d-aa incorporation. Single incorporation assays with four different tRNAs misacylated with six different l- and d-amino acids were performed in independent duplicates (triplicates for Lys). Template ‘G1’ was used for translation with tRNAGlyu (high EF-Tu and A-site affinity) and tRNAGlytp (low EF-Tu affinity, high A-site affinity) and template ‘O’ for tRNATyru (low EF-Tu and A-site affinity) and tRNATyrtp (high EF-Tu affinity, low A-site affinity). The anticodon on both tRNATyr was changed to UCA to enable opal stop codon suppression. (A) Autoradiograms of the gels. Remarkable differences in the d-aa incorporation efficiencies depending on EF-Tu affinity become apparent. (B) Signal intensities normalized to the respective reaction with l-aa-tRNAGlyu (lane 2 in (A)) are plotted. Control signals were subtracted from d-aa reaction signals prior to normalization. For clarity, the y-axis scaling is compressed above 150%. Error bars show the range of determined data.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499158&req=5

Figure 4: The effect of tRNA properties on d-aa incorporation. Single incorporation assays with four different tRNAs misacylated with six different l- and d-amino acids were performed in independent duplicates (triplicates for Lys). Template ‘G1’ was used for translation with tRNAGlyu (high EF-Tu and A-site affinity) and tRNAGlytp (low EF-Tu affinity, high A-site affinity) and template ‘O’ for tRNATyru (low EF-Tu and A-site affinity) and tRNATyrtp (high EF-Tu affinity, low A-site affinity). The anticodon on both tRNATyr was changed to UCA to enable opal stop codon suppression. (A) Autoradiograms of the gels. Remarkable differences in the d-aa incorporation efficiencies depending on EF-Tu affinity become apparent. (B) Signal intensities normalized to the respective reaction with l-aa-tRNAGlyu (lane 2 in (A)) are plotted. Control signals were subtracted from d-aa reaction signals prior to normalization. For clarity, the y-axis scaling is compressed above 150%. Error bars show the range of determined data.
Mentions: Since a full-length product band may arise from unspecific readthrough of a hungry codon or due to small enantiomeric impurities found in the d-aa derivatives (l-aa content <0.1–0.96%), each reaction was accompanied by a control dedicated to show the maximum possible extent of unspecific full-length product formation. The control reactions contained the same absolute amounts of tRNAGlyu, precisely deacyl-tRNAGlyu and 1% of the respective l-aa-tRNAGlyu to simulate the worst-case enantiomeric impurity. For evaluation, we assessed the intensities of the full-length product bands and subtracted the control signals from the d-aa signals, which we then related to the l-aa signals (Figure 3). Despite the relatively high variance between replicates, the data clearly point into the same direction: d-amino acids can reproducibly be incorporated with a reasonable efficiency. The variance is related to the fact that each data point results from two independent aminoacylation reactions (charging of l- and d-amino acid) and three independent translation reactions (control, l-aa, d-aa), followed by purification and analysis. Replicates were in no case conducted in parallel.

Bottom Line: We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases.The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation.Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.

View Article: PubMed Central - PubMed

Affiliation: NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany jachenbach@noxxon.com.

Show MeSH
Related in: MedlinePlus