# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 8 Inhibition of Translation by the Stringent Response  
In addition to controlling the transcription of rRNA and ribosome subunit assembly, (p)ppGpp also inhibits translational GTPases that are involved in the sequential process of protein production, namely bacterial initiation factor 2 (bIF-2), elongation factor Tu (EF-Tu) and recycling factor 3 (RF3) [118; 144; 145; 146]. Once the mature 30S subunit is assembled, mRNA can bind to bS1 and, thus, associate with the 30S subunit. bIF-2 is recruited, which enables association of the initiator fMet-tRNAMet to form Watson-Crick base pairing interactions between the mRNA start codon and the cognate tRNA anticodon. bIF-1 and bIF-3 facilitate bIF-2 and mRNA-30S binding, respectively [79], to form the 30S pre-IC. The 50S subunit then associates, with bIF-2 dissociating to remove the steric hindrance to 70S formation [147]. bIF-2 can bind to ppGpp in an identical fashion to GDP, maintaining the same hydrogen bonding networks [148]. It is well documented that bIF-2 can associate to the 30S pre-IC with high affinity in the GTP-bound state, but not while bound to GDP [145]. When interacting with (p)ppGpp, association of the fMet-tRNAMet to the 30S pre-IC is negatively affected, with the rate of the initial dipeptide bond formation decreasing [148].  
Following the formation of the 30S pre-IC and initiation of transcription, EF-Tu is responsible for escorting the aminoacyl-tRNAs to the A-site of the ribosome in preparation for residue incorporation [149]. Upon the correct situation of the tRNA, the GTPase activity of EF-Tu is activated and release from the A-site occurs. Subsequent association of EF-G close to the A-site provides energy to translocate mRNA and tRNA through the canonical and hybrid states while maintaining frame. Furthermore, recent structural studies have revealed that EF-G may in fact restrain the A-site tRNA, while maintaining the codon:anticodon interaction [150]. Both of these highly conserved and essential elongation factors bind to, and are inhibited by, ppGpp [151]. Indeed, EF-Tu can be inhibited either by direct competitive binding or by binding of ppGpp to a complex of EF-Tu and the cognate guanosine exchange factor EF-Ts to halt the EF-Tu cycle through kinetic trapping of this GTPase in an inactive complex [151]. Competitive inhibition of EF-G by ppGpp is also likely, as the use of an in vitro translation reconstitution system with a twofold excess of ppGpp over GTP, where EF-G was the limiting factor, revealed a twofold reduction in elongation rate [151].  
Finally, upon tRNA binding at the ribosomal exit site, the release of the tRNA is catalysed by class 1 release factors including RF-1 and RF-2 following GTP hydrolysis. The class 2 release factor RF-3 is pre-associated with the ribosome and is responsible for catalysing the release of RF-1 and RF-2 to facilitate the next round of elongation [152; 153]. Free RF-3 in the cytoplasm exists almost exclusively in the GDP-bound state, with a marked increase in ribosomal affinity when bound to GTP. In the ppGpp-bound state, it is hypothesised that the interaction of RF-3 with the ribosome would be negatively affected, supported by the fact that the recycling activity of RF-1 in a reconstituted assay decreased upon introduction of ppGpp into the system [153]. Furthermore, the reduction in the rate of exit of even a single tRNA can negatively influence the rate of translation of an entire polysome [154], suggesting that a slight reduction in RF-1 recycling could be more detrimental to total cellular translation than the reconstitution data suggests.  
Overall, the cumulative inhibition of formation of the 30S pre-IC, aminoacyl-tRNA recruitment through inhibition of EF-Tu, translocation of tRNA through inhibition of EF-G and tRNA release and Type-1 release factor recycling through binding to RF3 results in a pronounced negative effect on the rate of translation upon induction of the stringent response.