Title: Examining the Effects of D-Amino Acids on Translation
Description: The ribosome is responsible for mRNA-templated protein translation in all living cells. The translational machinery (TM) has evolved to use 20 amino acids each esterified onto one of several tRNA bodies. While the active site of the ribosome, known as the peptidyl transferase center (PTC), is able to handle a remarkable amount of substrate diversity, many classes of unnatural amino acids are not compatible with the TM. For example, in the field of unnatural amino acid mutagenesis, the site-specific incorporation of biologically useful amino acids into proteins, such as fluorophores, has often proven to be unfeasible. This runs counter to the accepted notion that the ribosome is blind to the structure of the amino acid and is capable of accepting any amino acid as long as the mRNA codon: tRNA anticodon pairing is correct. Two studies by our group set out to test the hypothesis that the ribosome can indeed discriminate the structure of the amino acid. Using a fully purified E. coli translation system, the first study showed that natural amino acids misacylated onto fully modified but non-native tRNAs show small but reproducible effects on the steps of aminoacyl-tRNA (aa-tRNA) selection.

The second study, in which I participated, utilized D-aa-tRNAs in the same E. coli translation system to study how amino acids of the inverted stereochemistry to those found in ribosomally-synthesized proteins affect translation elongation. We showed that these unnatural substrates serve as peptidyl acceptors but once translocated into the P-site of the ribosome, fail as peptidyl donors and stall translation elongation by inactivating the PTC. The motivation of my work has been to further characterize the effects of D-aa-tRNAs on translation elongation. To this end, I examined how the PTC is affected structurally and functionally by the presence of ribosomal substrates containing D-amino acids. Chapter one contains an introduction to this work. Chapter two describes chemical probing experiments that demonstrate that the presence of peptidyl-D-aminoacyl-tRNAs in the P-site of the ribosome allosterically modulates the secondary structure of ribosomal exit tunnel nucleotides A2058 and A2059.

Chapter three describes how the reactivity of peptidyl-D-aminoacyl-tRNAs to form tripeptides is highly dependent on the identity of the amino acid it is reacting with; protein yields can be close to what is obtained with natural amino acids or almost completely abolished. Chapter four contains the methods used to do this research. From the observations presented here as well as from the work of other laboratories, a picture of the PTC emerges in which the pairing of the A- and P- site substrates is integral in either promoting or suppressing catalysis by the PTC. This work has implications for the field of unnatural amino acid mutagenesis, particularly for strategies to improve the incorporation of interesting unnatural amino acid by the ribosome. In addition, this work adds an important aspect to the growing body of knowledge of ribosome stalling at the PTC.