Despite nearly 85 years of successful antibacterial chemotherapy, bacterial infections still pose a serious threat to human health. Continually emerging drug-resistant bacteria make existing agents less effective, and a paucity of new agents and decreased development effort from the pharmaceutical industry provide little hope of replenishing the arsenal (1, 2). Complications and mortality due to bacterial infections continue to increase, already reaching epidemic proportions in some areas of the world. If humans are to regain the upper hand in fighting bacterial infections, then innovation, investment, and new antibacterial agents will be needed. Although some of the barriers to the discovery and approval of new compounds are economic or policy related, there is also a desperate need for new targets and new mechanisms of action. A renewed effort to expand our knowledge of bacterial physiology and to translate discoveries into the clinic will be needed to address these challenges and to reinvigorate antibiotic development pipelines.Recent advances in our understanding of how bacteria maintain physiological homeostasis revealed a promising class of potential antibiotic targets called riboswitches-noncoding mRNAs that form a structured receptor (or aptamer) which can directly bind to a specific small-molecule ligand or ion and thereby regulate gene expression (3-5). Ligand binding to a riboswitch aptamer stabilizes a conformationally distinct architecture in the mRNA that modulates the expression of the adjacent coding region(s) (4-8). To date, more than 35 riboswitch classes have been discovered and characterized (7). Three of these riboswitch classes have been revealed as important cellular targets of antibacterial small molecules whose mechanism of action had not been previously defined (9-13). More recently, several publications have demonstrated that novel small molecules that bind to selected riboswitch aptamers with affinities comparable to that of the cognate ligand can be rationally identified and optimized (14-21).In some cases, synthetic or natural riboswitch ligand analogs have demonstrated potent antibacterial activity (12-15, 20, 22). For example, the phosphorylated form of roseoflavin (RoF) (

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR,B chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define-binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301.

We report here that tyrosyl-tRNA synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA synthetase and aligns with the prokaryotic tyrosyl-tRNA synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region. The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr). Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices. However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair. We show that P. carinii tyrosyl-tRNA synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr. In contrast, the enzyme does not charge E. coli microhelixTyr. Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P. carinii tyrosyl-tRNA synthetase. Conversely, we found that E. coli tyrosyl-tRNA synthetase can charge an E. coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair. The results demonstrate that the common structural framework of homologous tRNA synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation. We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA synthetases.

The trinucleotide/amino acid relationships of the present-day genetic code are established by the aminoacylation reactions of tRNA synthetases, whereby each of 20 specific amino acids is attached to its cognate tRNAs, which bear anticodon trinucleotides. Because of its universality, the appearance of the modern genetic code is thought to predate the separation of prokaryotic and eukaryotic organisms in the universal phylogenetic tree. In the light of new sequence information, we present here a phylogenetic analysis that shows an unusual picture for tyrosyl-and tryptophanyl-tRNA synthetases. In particular, the eukaryotic tyrosyl-and tryptophanyl-tRNA synthetases are more related to each other than to their respective prokaryotic counterparts. In contrast, each of the other 18 eukaryotic synthetases is more related to its prokaryotic counterpart than to any eukaryotic synthetase specific for a different amino acid. Our results raise the possibility that present day tyrosyl-and tryptophanyl-tRNA synthetases appeared after the separation of nucleated cells from eubacteria. The results have implications for the development of the genetic code.An open question concerning the origin of life is the mechanism of development of the genetic code and the role played by aminoacyl-tRNA synthetases (1). Because of their central role in linking amino acids with nucleotide triplets contained in transfer RNAs (2-6), aminoacyl-tRNA synthetases are thought to be among the first proteins that appeared in evolution. In agreement with this view of an ancient origin for the enzymes, all phylogenetic analyses carried out so far have determined that the sequences of tRNA synthetases cluster together by their amino acid specificities, and not by their positions in the phylogenetic tree (7-9). This observation clearly indicates that the enzymes appeared and evolved into their current types before the branching of the three Urkingdoms (eubacteria, archaebacteria, and eukaryotes). These previous analyses, however, were limited by the lack of (mainly) eukaryotic sequences for some of the enzymes. For instance, no eukaryotic tyrosyl-tRNA synthetase (YRS) or tryptophanyl-tRNA synthetase (WRS) had been analyzed. Here we present an analysis of the phylogenetic relationships between the prokaryote and eukaryote forms of this pair of enzymes.Recent determination by Carter and coworkers (10) of the crystal structure of a eubacterial WRS (B-WRS) revealed a high degree of structural similarity between this enzyme and the eubacterial YRS (B-YRS) solved by Brick et al. (11), thus suggesting a surprisingly recent common ancestor not discernible through sequence analysis. This structural conservation is particularly remarkable considering the vast amount of time since both of these class I tRNA synthetases separated from their ancestor. No three-dimensional information is available for any eukaryotic WRS or YRS (E-WRS or E-YRS).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...