Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a “superlattice” of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

Integrins play myriad and vital roles in development and disease. They connect a cell with its surroundings and transmit chemical and mechanical signals across the plasma membrane to the cell’s interior. Dissecting their roles in cell behavior is complicated by their overlapping ligand specificity and shared downstream signaling components. In principle, synthetic peptides can be used to modify surfaces to mimic extracellular matrix proteins by supporting integrin-mediated adhesion, but most short peptide sequences lack selectivity for one integrin over others. In contrast, synthetic integrin antagonists can be highly selective. We hypothesized that this selectivity could be exploited if antagonists, when immobilized, could support cellular adhesion and activate signaling by engaging specific cell-surface integrins. To investigate this possibility, we designed a bifunctional (RGD)-based peptidomimetic for surface presentation. Our conjugate combines a high affinity integrin ligand with a biotin moiety; the former engages the αvβ3 integrin and the latter allows for presentation on streptavidin-coated surfaces. Surfaces decorated with this ligand promote both cellular adhesion and integrin activation. Moreover, the selectivity of these surfaces for the αvβ3 integrin can be exploited to capture a subset of cells from a mixed population. We anticipate that surfaces displaying highly selective small molecule ligands can reveal the contributions of specific integrin heterodimers to cell adhesion and signaling.

An efficient asymmetric synthesis of the 22nd amino acid L-pyrrolysine has been accomplished. The key stereogenic centers were installed by an asymmetric conjugate addition reaction. A Staudinger/aza-Wittig cyclization was used to form the acid-sensitive pyrroline ring. Pyrrolysine was synthesized in thirteen steps in 20% overall yield.