In a living cell, peptides or proteins produced by ribosomal translation of a messenger RNA consist exclusively of L-amino acids (L-aa), although D-amino acids (D-aa) are prevalent in nature. The bacterial translation apparatus holds several mechanisms to counteract the incorporation of D-aa into peptides or proteins by ribosomal synthesis, because the accidental incorporation of D-aa may hamper the folding of a nascent protein into its active conformation. Accumulation of such misincorporations would have lethal consequences for a cell. The chirality of the protein building blocks is controlled at four sequent checkpoints: 1. Aminoacyl-tRNA synthetases (aaRS) esterify amino acids to tRNA molecules, giving rise to aminoacyl-tRNA. Most of the enzymes are stereoselective and do not charge D-amino acids. 2. D-tyrosyl-tRNA deacylase controls the formed aminoacyl-tRNAs and selectively deacylates D-amino acids. 3. Elongation factor Tu (EF-Tu) is a translation factor that binds to aminoacyl-tRNA and shuttles it to the ribosome, but strongly discriminates against D-aminoacyl-tRNA. 4. The ribosome uses aminoacyl-tRNA as a substrate to build up peptide chains in an mRNA sequence dependent manner. The ribosome is also stereoselective and can reject D-aminoacyl-tRNA. This work focuses on the bypassing of stereoselectivity of the E. coli translation apparatus to achieve efficient site-directed incorporation of D-amino acids, which can be useful both for a number of applications such as drug discovery by in vitro translation based techniques as well as to deepen the understanding how stereoselectivity is established on a molecular level. This goal has been achieved by the following means: 1. D-amino acids are attached to tRNA molecules using ribozymes (Flexizymes) instead of aminoacyl-tRNA synthetases. 2. A defined cell-free in vitro translation system made of individually purified translation factors and ribosomes or mutants thereof was prepared that does not contain D-tyrosyl-tRNA deacylase. 3. To enable EF-Tu mediated delivery of D-aa-tRNA to the ribosome, the D-aa were attached to a tRNA that shows a high intrinsic affinity for EF-Tu. 4. The equilibrium between D-aa-tRNA rejection and D-aa incorporation by the ribosome was shifted towards incorporation by using a tRNA that also shows a high intrinsic affinity to the ribosomal A-site. The efficient incorporation of 17 different D-aa out of 18 D-aa tested could be demonstrated and in some cases also the incorporation of consecutive D-aa.
|School:||Technische Universitaet Berlin (Germany)|
|Source:||DAI-C 81/1(E), Dissertation Abstracts International|
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