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Ribonucleotide modifications perform a wide variety of roles in synthesis, turnover and functionality of tRNA molecules. The presence of particular chemical moieties can refine the internal interaction network within a tRNA molecule, influence its thermodynamic stability, contribute novel chemical properties and affect its decoding behavior during mRNA translation. As the lack of specific modifications in the anticodon stem and loop causes disrupted proteome homeostasis, diminished response to stress conditions, and the onset of human diseases, the underlying modification cascades have recently gained particular scientific and clinical interest. Nowadays, a complicated but conclusive image of the interconnectivity between different enzymatic modification cascades and their resulting tRNA modifications emerges. Here we summarize the current knowledge in the field, focusing on the known instances of cross talk among the enzymatic tRNA modification pathways and the consequences on the dynamic regulation of the tRNA modificome by various factors. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

tRNA genes are transcribed by RNA polymerase III (RNAPIII). During recent years it has become clear that RNAPIII activity is strictly regulated by the cell in response to environmental cues and the homeostatic status of the cell. However, the molecular mechanisms that control RNAPIII activity to regulate the amplitude of tDNA transcription in normally cycling cells are not well understood. Here, we show that tRNA levels fluctuate during the cell cycle and reveal an underlying molecular mechanism. The cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to boost tDNA transcription during late S phase. At tDNA genes, Cdk1 promotes the recruitment of TFIIIC, stimulates the interaction between TFIIIB and TFIIIC, and increases the dynamics of RNA polymerase III in vivo. Furthermore, we identified Bdp1 as a putative Cdk1 substrate in this process. Preventing Bdp1 phosphorylation prevented cell cycle-dependent recruitment of TFIIIC and abolished the cell cycle-dependent increase in tDNA transcription. Our findings demonstrate that under optimal growth conditions Cdk1 gates tRNA synthesis in S phase by regulating the RNAPIII machinery, revealing a direct link between the cell cycle and RNAPIII activity.

The introduction of antibiotics for the chemotherapy of bacterial infections has been one of the most important medical achievements of the past 50 years. However, the emergence of bacterial resistance to antibiotics undermines the therapeutic utility of existing agents, creating a requirement for the discovery of new antibacterial drugs. Several drug discovery strategies have emerged, including incremental improvements to existing antibiotics by chemical manipulation and the search for novel drug targets based on genomic approaches. An alternative strategy seeks to exploit opportunities for drug discovery arising from an understanding of the mode of action of existing antibiotics. Thus biochemical pathways or processes inhibited by antibiotics already in clinical use may nevertheless contain key functions that represent unexploited targets for further drug discovery. A major benefit of employing pathways or processes that are already known to contain drug targets is that proof of principle for drug intervention is already established. This approach to drug discovery is illustrated by reviewing target sites for existing antibiotics and considering how this information might be applied for the discovery of new agents inhibiting peptidoglycan synthesis, tRNA synthesis, transcription and DNA replication

A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure–activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC50s of 21.7 and 35 μM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 μM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

The mechnisms of synthesis of mammalian selenocysteyl-tRNA were studied with murine active enzyme fractions. (75Se)Selenocysteyl-tRNA was synthesized in vitro by 500kDa protein from seryl-tRNA and (75Se)HSe-, which should be activated with ATP by 20kDa protein. This and other factors in mamalian systems are compared with those in Escherichia coli.

The mechnisms of synthesis of mammalian selenocysteyl-tRNA were studied with murine active enzyme fractions. (75Se)Selenocysteyl-tRNA was synthesized in vitro by 500kDa protein from seryl-tRNA and (75Se)HSe-, which should be activated with ATP by 20kDa protein. This and other factors in mamalian systems are compared with those in Escherichia coli.

Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to tRNA by the aa-tRNA synthetases. An exception is Gln-tRNA synthesis, which in eukaryotes is catalyzed by glutaminyl-tRNA synthetase (GlnRS), while most bacteria, archaea, and chloroplasts employ the transamidation pathway, in which a tRNA-dependent glutamate modification generates Gln-tRNA. Mitochondrial protein synthesis is carried out normally by mitochondrial enzymes and organelle-encoded tRNAs that are different from their cytoplasmic counterparts. Early work suggested that mitochondria use the transamidation pathway for Gln-tRNA formation. We found no biochemical support for this in Saccharomyces cerevisiae mitochondria, but demonstrated the presence of the cytoplasmic GlnRS in the organelle and its involvement in mitochondrial Gln-tRNA synthesis. In addition, we showed in vivo localization of cytoplasmic tRNAGln in mitochondria and demonstrated its role in mitochondrial translation. We furthermore reconstituted in vitro cytoplasmic tRNAGln import into mitochondria by a novel mechanism. This tRNA import mechanism expands our knowledge of RNA trafficking in the eukaryotic cell. These findings change our view of the evolution of organellar protein synthesis.

Transfer RNA is primarily synthesized by transcription by RNA polymerase and becomes the mature form via several steps: processing, splicing, CCA addition and posttranscriptional modification. The concentration of individual tRNA molecules is controlled in order to maintain cellular functions. Keywords: codon usage; gene-dosage effect; promoter; modified nucleotides; tRNA processing

Some aspects of tRNA synthesis inAureobasidium pullulans were studied during its development in synthetic liquid medium. tRNA methylation detected by labeling withMe-14C-l-methionine was maximum at the beginning of conidiogenesis. The results suggest that tRNA plays an important role in differentiation processes.

Levels of transfer RNA (tRNA) were determined in liver, kidney, skeletal muscle, heart, and brain of young (35-day), adult (12-month) and old (24-month) female C57BL/6J mice. Kidney and liver showed little change in tRNA levels between young and adult mice, but the levels decreased in old mice. Skeletal muscle tRNA decreased steadily from young to old mice. Heart tRNA increased during maturation to adult organisms and then decreased in old individuals. Brain levels of tRNA increased steadily. No age-related change in the rate of transport of orotic acid into cells was observed. However, all tissues exhibited a decrease in uridine pools between adult and old mice. Most importantly, all tissues of aging mice showed a decrease in the rate of tRNA synthesis.

Strains of Saccharomyces cerevisiae have been constructed that possess temperature-sensitive defects in tRNA precursor (pre-tRNA) splicing and which also lack the processing endonuclease that acts at the 3′-terminus of 5 S rRNA and 35 S rRNA precursors (pre-rRNAs). The unspliced pre-tRNAs accumulated by such strains at the nonpermissive temperature are identical in structure to those accumulated by pre-tRNA splicing-defective strains with a functional pre-5 S RNA processing enzyme. The pre-RNA processing activity is therefore not obligatorily involved in maturation of several yeast tRNAs. However, gels of the pulse-labelled RNAs of RNA82+ and rna82.1 strains provide evidence that this enzyme acts upon a few small unstable transcripts that are not 5 S RNA forms. The most prominent of these transcripts on gels was, in wild-type strains, an RNA 145 ± 2 nucleotides in length.

The relative rates of tRNA, rRNA, and DNA synthesis were measured for steady state cultures of Escherichia coli B/r growing in succinate and glucose minimal media which support growth rates of 0.67 and 1.46 doublings per hour, respectively. Following a 2-fold increase in growth rate, the rate of tRNA synthesis increased approximately 2-fold relative to the rate of DNA synthesis and decreased 10 to 15% relative to the rate of rRNA synthesis. It is concluded that the synthesis of tRNA is regulated in a manner similar to the mechanism regulating rRNA synthesis. The 10 to 15% difference in the relative rates of synthesis is not the result of an increase in the relative frequency of ribosomal RNA cistrons at the higher growth rates, and therefore it seems that the mechanisms controlling rRNA and tRNA synthesis are at least partially independent.

Publisher Summary This chapter discusses the synthesis, chemistry, and structural features of hypermodified nucleosides of tRNA. It deals with the direct effects of hypermodification altering nucleoside base-pairing and stacking properties as a result of inherent electronic and conformational changes. A brief inspection of the anticodon loop structure of tRNA Phe suggests the possibility of some indirect effects resulting from the interactions with the anticodon loop backbone, such as steric repulsion of bulk substituents, electrostatic repulsion of ionized groups (carboxyl and phosphodiester), hydrogen bonding, and Mg 2+ binding. Lack of accurate information about the tertiary structures of different tRNAs makes the evaluation of such indirect effects rather speculative. Some information can be gleaned from the simulation studies in which hypermodified nucleosides in their crystal structures are built into the yeast tRNA Phe anticodon loop at the positions of Gm 34 and of yW37, with the assumption that at least the main structural features are preserved in all tRNAs.

Rat liver methylating enzymes could methylate tRNA extracted from the livers of rats treated with 35 mg/100 g L-ethionine 19 h prior to sacrifice. 1-methylhypoxanthine and 3-methylcytosine were among the methylated bases synthesized in vitro . The synthesis of 3-methylcytosine was dependent on the presence of Mg++ although this ion inhibited the overall methylation of the tRNA.

The effect of spermine on the rates of ATP-pyrophosphate exchange and of formation of threonyl-tRNA by threonyl tRNA synthetase has been studied. It is shown that spermine alone has no effect on the overall reaction as well as on the rate of ATP pyrophosphate isotope exchange. At low Mg2+ concentration spermine activates 8-fold the ATP-pyrophosphate isotope exchange and 23-fold the threonyl tRNA synthesis. The possible role of spermine in this activation is discussed.