Small-Molecule Antibiotics Inhibiting tRNA-Regulated Gene Expression Is a Viable Strategy for Targeting Gram-Positive Bacteria.

Bacterial infections and the rise of antibiotic resistance, especially multidrug resistance, have generated a clear need for discovery of novel therapeutics. We demonstrated that a small-molecule drug, PKZ18, targets the T-box mechanism and inhibits bacterial growth. The T-box is a structurally conserved riboswitch-like gene regulator in the 5' untranslated region (UTR) of numerous essential genes of Gram-positive bacteria. T-boxes are stabilized by cognate, unacylated tRNA ligands, allowing the formation of an antiterminator hairpin in the mRNA that enables transcription of the gene. In the absence of an unacylated cognate tRNA, transcription is halted due to the formation of a thermodynamically more stable terminator hairpin. PKZ18 targets the site of the codon-anticodon interaction of the conserved stem I and reduces T-box-controlled gene expression. Here, we show that novel analogs of PKZ18 have improved MICs, bactericidal effects against methicillin-resistant Staphylococcus aureus (MRSA), and increased efficacy in nutrient-limiting conditions. The analogs have reduced cytotoxicity against eukaryotic cells compared to PKZ18. The PKZ18 analogs acted synergistically with aminoglycosides to significantly enhance the efficacy of the analogs and aminoglycosides, further increasing their therapeutic windows. RNA sequencing showed that the analog PKZ18-22 affects expression of 8 of 12 T-box controlled genes in a statistically significant manner, but not other 5'-UTR regulated genes in MRSA. Very low levels of resistance further support the existence of multiple T-box targets for PKZ18 analogs in the cell. Together, the multiple targets, low resistance, and synergy make PKZ18 analogs promising drugs for development and future clinical applications.

Discovery of Small-Molecule Antibiotics against a Unique tRNA-Mediated Regulation of Transcription in Gram-Positive Bacteria.

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5'-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5'UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 µg mL-1 ). Resistance developed at an extremely low mutational frequency (1.21×10-10 ). At 4 µg mL-1 , the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd ≈24 µm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

Celebrating wobble decoding: Half a century and still much is new.

A simple post-transcriptional modification of tRNA, deamination of adenosine to inosine at the first, or wobble, position of the anticodon, inspired Francis Crick's Wobble Hypothesis 50 years ago. Many more naturally-occurring modifications have been elucidated and continue to be discovered. The post-transcriptional modifications of tRNA's anticodon domain are the most diverse and chemically complex of any RNA modifications. Their contribution with regards to chemistry, structure and dynamics reveal individual and combined effects on tRNA function in recognition of cognate and wobble codons. As forecast by the Modified Wobble Hypothesis 25 years ago, some individual modifications at tRNA's wobble position have evolved to restrict codon recognition whereas others expand the tRNA's ability to read as many as four synonymous codons. Here, we review tRNA wobble codon recognition using specific examples of simple and complex modification chemistries that alter tRNA function. Understanding natural modifications has inspired evolutionary insights and possible innovation in protein synthesis.

The Importance of Being Modified: The Role of RNA Modifications in Translational Fidelity.

The posttranscriptional modifications of tRNA's anticodon stem and loop (ASL) domain represent a third level, a third code, to the accuracy and efficiency of translating mRNA codons into the correct amino acid sequence of proteins. Modifications of tRNA's ASL domain are enzymatically synthesized and site specifically located at the anticodon wobble position-34 and 3'-adjacent to the anticodon at position-37. Degeneracy of the 64 Universal Genetic Codes and the limitation in the number of tRNA species require some tRNAs to decode more than one codon. The specific modification chemistries and their impact on the tRNA's ASL structure and dynamics enable one tRNA to decode cognate and "wobble codons" or to expand recognition to synonymous codons, all the while maintaining the translational reading frame. Some modified nucleosides' chemistries prestructure tRNA to read the two codons of a specific amino acid that shares a twofold degenerate codon box, and other chemistries allow a different tRNA to respond to all four codons of a fourfold degenerate codon box. Thus, tRNA ASL modifications are critical and mutations in genes for the modification enzymes and tRNA, the consequences of which is a lack of modification, lead to mistranslation and human disease. By optimizing tRNA anticodon chemistries, structure, and dynamics in all organisms, modifications ensure translational fidelity of mRNA transcripts.

Chemical and Conformational Diversity of Modified Nucleosides Affects tRNA Structure and Function.

RNAs are central to all gene expression through the control of protein synthesis. Four major nucleosides, adenosine, guanosine, cytidine and uridine, compose RNAs and provide sequence variation, but are limited in contributions to structural variation as well as distinct chemical properties. The ability of RNAs to play multiple roles in cellular metabolism is made possible by extensive variation in length, conformational dynamics, and the over 100 post-transcriptional modifications. There are several reviews of the biochemical pathways leading to RNA modification, but the physicochemical nature of modified nucleosides and how they facilitate RNA function is of keen interest, particularly with regard to the contributions of modified nucleosides. Transfer RNAs (tRNAs) are the most extensively modified RNAs. The diversity of modifications provide versatility to the chemical and structural environments. The added chemistry, conformation and dynamics of modified nucleosides occurring at the termini of stems in tRNA's cloverleaf secondary structure affect the global three-dimensional conformation, produce unique recognition determinants for macromolecules to recognize tRNAs, and affect the accurate and efficient decoding ability of tRNAs. This review will discuss the impact of specific chemical moieties on the structure, stability, electrochemical properties, and function of tRNAs.