## 2 Antibiotics Affecting tRNA Biogenesis and Modification  
### Antibiotics Preventing tRNA Maturation  
Ribonuclease P is an endoribonuclease that cleaves the 5'-end of pre-tRNA to generate mature tRNA. This is an essential step in the biogenesis of tRNA. A bacterial RNase P holozyme structure has been elucidated from _E. coli_ and comprises a catalytic RNA or ribozyme subunit (350 to 450 nucleotides), and a single protein subunit (110 to 150 amino acids) [15]. The RNA subunit is termed M1 and the protein subunit is referred to as C5. The interactions between M1 and C5 are essential for the recognition and binding to its substrates [16]. A comparison of the RNase P subunits between different bacteria indicates only some degree of identity. For example, there is only 30% similarity between the protein subunits of _Bacillus subtilis_ and _E. coli_. Importantly, from an antibiotic perspective, there exist striking differences between bacterial and human RNase P (which contains a much higher number of protein subunits). These differences between host and microbe make RNase P an attractive candidate for the design of new antimicrobials. A number of RNase P targeting antibiotics are known, which use a number of different modes of action. Inhibition mechanisms include: Binding to the pre-tRNA substrate and disrupting RNase P substrate recognition; binding to RNase P itself and altering the P RNA conformation or disrupting the association of RNA and protein in the complex; and finally binding and disruption of the enzyme-substrate complex (RNase P-pre-tRNA). Some examples of antibiotics that bind to pre-tRNA substrates include aminoglycos, which displace functional metal ions, and have been shown to prevent _E. coli_ RNase P recognizing the substrate [17, 18, 19, 20]. Specifically, neomycin B binds pre-tRNA with a half maximal inhibitory concentration (IC\({}_{50}\)) of 60 \(\upmu\)M) [21] (Figure 2); While derivatives of neomycin with positively charged lysine, arginine or guanidinium groups exhibit IC\({}_{50}\) values ranging from 0.1 to 6 \(\upmu\)M [18, 19]. _E. coli_ RNase P inhibitors porphines and porphyrins with inhibition constant (\(K_{\mathrm{i}}\)) ranging from 1 to 4 \(\upmu\)M work in a similar manner displacing metal ions and/or by altering the tertiary structure of tRNA [20, 22, 23]. Synthetic RNase P inhibitors, such as bis-benzimidazoles have been found to bind to the T-stem of _E. coli_ pre-tRNA\({}^{\mathrm{Phe}}\) and prevent RNase P interaction (IC\({}_{50}\) of 5-20 \(\upmu\)M) [24]. Interestingly, peptidyl-transferase inhibitors (e.g., puromycin (IC\({}_{50}\)-3 mM); Amicetin and blasticidin S) can also block RNase P activity by binding to pre-tRNA [25, 26]. Antisense technology has also been utilized to target the RNA moiety of RNase P itself [27] with inhibitors towards _E. coli_ and _B. subtilis_ RNase P with IC\({}_{50}\) values obtained of 2 nM to 1 \(\upmu\)M, respectively [27, 28]. The P15 loop region of _E. coli_ RNase P is important in forming interactions with the 3'-end of the pre-tRNA substrate. [PERSON] _et al._, synthesized a 14 base oligomer that is stable and resistant to nucleases and demonstrated that it binds irreversibly to RNase P. These _in vitro_ studies showed that the antisense oligomer specifically targets the catalytic core (P15 loop) of RNase P and traps it in a partially unfolded state, thereby affecting substrate binding. In addition to this it also affects the coordination of Mg\({}^{2+}\) ions that are important for catalysis. The success of this technique is due to the accessibility of the oligomer to this P15 loop region. Further investigations of this oligonucleotide _in vivo_ conjugated it to an invasive peptide via a monoglycine linker (peptide nucleic acid) to facilitate the uptake of the inhibitor into live _E. coli_ cells. Inhibition of growth was observed indicating a successful uptake of the oligomer by the cells and specific targeting of the _E.