# The Impact of the Stringent Response on TRAF GTPases and Prokaryotic Ribosome Assembly
## 4 (p)ppGpp-Binding RA-GTPases Involved in 30S Assembly
### Era  
Era is a highly conserved protein across prokaryotes and eukaryotes with pleiotropic roles within the cell, including ribosome assembly, cell cycle control and apoptosis [69; 70; 71]. Era is a two-domain protein featuring a C-terminal KH RNA-binding domain as well as the N-terminal G domain (Figure 2). The KH domain is responsible for binding to the 3\({}^{\prime}\) end of the 16S rRNA adjacent to the anti-Shine-Dalgarno (SD) sequence (Figure 3) [72]. Here it recognises the GAUCA motif, which along with h45 (nucleotides 1506-1529) is universally conserved [73]. The crystal structure of Era bound to GMPPNP exhibits a closed domain structure in which the G and KH domains close around the ligand, whereas in the apo and GDP-bound states the domain structure is open [61]. This displacement covers a range of approximately 10-15 A and may suggest that Era binding to the 30S depends on the bound nucleotide. In the mature 30S this binding site is occupied by b51--the final r-protein in the 30S assembly pathway; Era and b51 from _E. coli_ cannot coexist on the pre-30S particle [59]. While the GAUCA motif is crucial for Era binding to the 16S, optimum GTPase stimulation is reliant on the adjacent anti-SD sequence (CCUCC), so altogether the correctly matured 1530 GAUCACCUCC1539 region is required for up to 12-fold stimulation of Era activity [73; 74]. It has been determined experimentally that Era binding to the ribosomal subunit interface can prevent 30S and 50S joining, indicating that Era is important as a checkpoint for 16S maturity prior to 70S assembly [75]. Indeed, deleting _era_ in _Staphylococcus aureus_ caused a marked decrease in 70S formation, with an increase in mature 50S subunits relative to 30S, indicating a 30S processing defect [76]. Cryo-EM data from strains with controlled _era_ depletion in _E. coli_ accumulate 30S subunit assembly intermediates ranging from late-stage to early-stage, and also reveal that the 30S platform region remains unfolded in the absence of Era, with densities lacking for u52, u5S and b521 [77]. Similarly, the density maps of h23 and h24 are highly fragmented and h44 and h45 were completely absent [77]. The authors thus suggest that the folding of h23 and h24 is a major kinetic barrier in the correct maturation of the 30S subunit, especially the head region, and that Era facilitates the efficacy of this process [77]. It has been shown that, during 30S pre-initiation complex (30S pre-IC) formation, IF3 binds to the 16S rRNA at nucleotides 1532-1534 within the GAUCA motif [78]. The 30S pre-IC is a complex of IF1, IF2, IF3, mRNA and N-formylmethionine (fMet)-tRNA, which assembles prior to 70S formation and translational initiation and is an absolute requirement for 30S-50S joining into a translating 70S particle [79]. Mutation of 1530 GA1531 also affects IF3 association via alteration of the 16S 3\({}^{\prime}\) conformation, which differs from the Era-bound and IF3-bound states [80]. Due to the overlap of Era and IF3 binding sites, it appears that Era occludes IF3 binding and 30S pre-IC formation while bound to the 16S. Taken together with the fact that Era binding also occludes the anti-SD sequence and, thus, prevents mRNA recruitment to the 30S pre-IC, it is feasible that Era-mediated 16S maturation is in actuality the final stage of 30S assembly before the initiation of translation. A number of studies using mutant bacterial strains also shed light on the cellular function of Era. This protein is essential for growth in _E. coli_, although can be deleted in _S. aureus_[76; 81; 82; 83]. Overexpression Era can partially suppress the phenotypes present in \(\Delta r\underline{s}A\) mutants, indicating an overlap of function [54; 66; 76]. Era may however, function downstream of RsgA, given that a 20-fold excess of Era increases the dissociation constant of RsgA binding to the 30S from 58.5 nM to 2.3 nM through destabilisation of h44 [77].