| Summary: | Modified bases in RNA molecules are common, with over one-hundred individual modifications reported to date. In transfer RNA (tRNA), these modifications help hold the molecule in its proper conformation, allow the aminoacyl-synthetases to attach the amino acid to the correct tRNA for transfer to the ribosome, and in the correct matching of the codon-anticodon pair at the ribosome. Two positions on the tRNA molecule are significant in this last action; positions 34 and 37. Position 34 is the first base in the anticodon, and pairs with the last base in the codon during translation; modifications at this point allow the anticodon to pair with multiple codons. Modifications at position 37 help stabilise the anticodon loop of the tRNA, and help the efficiency of the translation. The threonylcarbamyoladenosine (t<sup>6</sup>A) modification at position 37 is required for the efficient translation of ANN codons. At least four enzymes, TsaBCDE, are involved in the formation of this modification, and are essential for bacterial viability. In this study, the interactions between the TsaB, TsaC, TsaD and TsaE have been investigated. Protein complex formation has been identified both in-vitro and in-vivo between TsaB and TsaD. The proteins interact in a 1:1 ratio, and the results indicate that this interaction stabilises the TsaD enzyme. The interaction appears to be unchanged over a physiological range of temperatures. No stable complex was identified between TsaC or TsaE with any of the other enzymes, however calorimetry suggests that there is an interaction between TsaE and both TsaB and TsaD. TsaC is believed to synthesise the threonylcarbamoyladenylate intermediate from threonine, bicarbonate and ATP; the activity of the enzyme complex (as measured by ATPase activity) in a media containing these compounds is increased in the presence of TsaC. If the cell is prevented from maintaining a source of t<sup>6</sup>A, it mounts a number of transcriptional responses. The transcription of certain amino acid biosynthetic genes is increased, corresponding to the tRNAs requiring the t<sup>6</sup>A modification, and in the transport and conversion of sulphates. This reflects the cellular response to starvation of an amino acid; the inability to incorporate the amino acids into the polypeptide chain due to the hypomodified tRNA is interpreted as the absence of that particular amino acid. Other transcriptional effects can be interpreted as a consequence of translational stalling where the t<sup>6</sup>A modification is required, leading to alterations in transcriptional promotion. TsaB and TsaD have also been shown to bind a small number of compounds in a high-throughput fragment-based drug discovery assay. Some of these have been shown to potentially affect the ATPase activity of the enzymes.
|
|---|
