The cellular translational machinery (TM) synthesizes proteins using exclusively L-or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aatRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.A lthough the ribosome catalyzes protein synthesis using more than 20 chemically diverse natural amino acids, these natural amino acids are all of L-or achiral configurations. As a consequence, the ability of the ribosome to incorporate L-aminoacyltRNAs (L-aa-tRNAs) with both a high degree of speed and accuracy has been the focus of decades of intense mechanistic and structural investigations (1-3). In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8...

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The involvement of tRNA structural elements beyond the anticodon in aminoacyl-tRNA (aa-tRNA) selection by the ribosome has revealed that substrate recognition is considerably more complex than originally envisioned in the adaptor hypothesis. By combining recent breakthroughs in aa-tRNA synthesis and mechanistic and structural studies of protein synthesis, we ask if aa-tRNA recognition further extends to the amino acid, thereby explaining various translation disorders exhibited by misacylated tRNAs. Contrary to expectation, we find that natural amino acids misacylated onto natural, but non-native tRNAs are selected with efficiencies very similar to those of their correctly-acylated counterparts. Despite this, small, but reproducible differences in selection indeed demonstrate that the translational machinery is sensitive to the amino acid/tRNA pairing. These results suggest that either the ribosome is an exquisite sensor of natural versus unnatural amino acid/tRNA pairings and/or that aa-tRNA selection is not the primary step governing the amino acid specificity of the ribosome.

Painful burn injuries are among the most debilitating form of trauma, globally ranking in the top 15 leading causes of chronic disease burden. Despite its prevalence, however, chronic pain after burn injury is under-studied. We previously demonstrated the contribution of the Rac1-signaling pathway in several models of neuropathic pain, including burn injury. However, Rac1 belongs to a class of GTPases with low therapeutic utility due to their complex intracellular dynamics. To further understand the mechanistic underpinnings of burn-induced neuropathic pain, we performed a longitudinal study to address the hypothesis that inhibition of the downstream effector of Rac1, Pak1, will improve pain outcome following a second-degree burn injury. Substantial evidence has identified Pak1 as promising a clinical target in cognitive dysfunction and is required for dendritic spine dysgenesis associated with many neurological diseases. In our burn injury model, mice exhibited significant tactile allodynia and heat hyperalgesia and dendritic spine dysgenesis in the dorsal horn. Activity-dependent expression of c-fos also increased in dorsal horn neurons, an indicator of elevated central nociceptive activity. To inhibit Pak1, we repurposed an FDA-approved inhibitor, romidepsin. Treatment with romidepsin decreased dendritic spine dysgenesis, reduced c-fos expression, and rescued pain thresholds. Drug discontinuation resulted in a relapse of cellular correlates of pain and in lower pain thresholds in behavioral tests. Taken together, our findings identify Pak1 signaling as a potential molecular target for therapeutic intervention in traumatic burn-induced neuropathic pain.