## 4 The Stringent Response  
Beyond the direct role in translation, tRNA molecules are involved in multiple metabolic and cellular routines [14,197,198]. Recent data also indicate that a mitochondrial tRNA is part of the architectural framework of the large mitochondrial ribosomal subunit (39S) itself, replacing the role of bacterial 5S rRNA as an organizing scaffold of the central protuberance region [199]. In the following section, we will focus on one important example of such a metabolic process, the induction of the stringent response by uncharged tRNAs, which serve as sensors for starvation and thus, are part of the regulation of global gene expression. Living organisms face fast environmental changes upon which they have to adjust quickly and accurately in order to survive. Such important signaling pathways exist in all kingdoms of life, those related to modified nucleotides being the more prominent ones [200]. The bacterial stringent response, first identified due to the inability of certain _E. coli_ mutants to reduce the amount of RNA synthesis in starvation conditions [201], is triggered during amino acid starvation (nutritional stress) and uses the alarmone (p)ppGpp (a hyper-phosphorylated version of GDP or GTP) to modulate a wide range of genes via transcription repression [202, 203, 204]. The factor responsible for sensing and producing the effective response, _i.e._, the synthesis of the signaling molecule (p)ppGpp, is a ribosome activated enzyme termed RelA. Decades of biochemical and genetic studies have provided a detailed picture on how and when (p)ppGpp is produced in the cell and the stringent response activated. When bacterial cells are growing in exponential phase, only \(\sim\)15% of all cellular tRNAs are deacylated [205], but most of them are engaged in regular cellular processes or ready to be re-aminoacylated. On the contrary, when the environmental conditions suffer a drastic change and there is scarcity of free amino acids, the concentration of non-acylated-tRNAs increases drastically, being able to interfere with the translation cycle by competing with the EF-Tu-GTP-aa-tRNA\({}^{\rm aa}\) ternary complexes to occupy the vacant A site of the ribosome. In this scenario, the peptide transfer from the tRNA in the P site to the tRNA on the A site cannot be performed and the ribosomes are stalled with cognate, non-acylated tRNA on the A site. This is the molecular event sensed by RelA, which binds to the stalled ribosomes to catalyze the transfer of the \(\beta\) and \(\gamma\) phosphates from ATP to GTP or GDP [206]. A cryo-EM structure of the stringent factor RelA bound to translating but stalled ribosomes with cognate, deacylated tRNA species in the ribosomal A site was recently determined for the first time [207]. The structures revealed that RelA binds the large ribosomal subunit via L11 and the 30S subunit though interactions established with the shoulder domain of the 16S rRNA. Moreover, the structure showed that the deacylated tRNA is distorted into an A/T like state to interact with RelA, a configuration that enables the recognition of the deacylation state of the tRNA species as it exposes its 3' end. This pseudo A/T conformation is reminiscent of the one induced by the ternary complex with acylated-tRNAs during the incorporation of cognate tRNA species to the A site in a standard elongation cycle (Figure 9). The novel conformation of the tRNA allows the proposition of a mechanism by which RelA might be activated, as it might serve as a scaffold to nucleate the catalytic 3D conformation of the factor. In a first step, the initial binding of the stringent factor to the stalled ribosome proceeds through the shoulder region of the small subunit, likely via the _C_-terminal region of RelA, which represents the regulatory and ribosome-binding domain [208, 209].