Outwitting EF-Tu and the ribosome: translation with d-amino acids.
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.
Affiliation: NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany firstname.lastname@example.org.Show MeSH
Related in: MedlinePlus
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.
Affiliation: NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany email@example.com.