# Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria
## 2 30S Ribosomal Subunit Biogenesis Factors
### Era  
The ribosome biogenesis factor Era belongs to the Ras-like small GTPase family (_E. coli_Ras-like) [50]. Ras is an acronym for rat sarcoma, in which the first members of the Ras family were discovered [51]. Era is a vital GTP-binding protein found in almost all bacteria [52; 53; 54; 55; 56]. Era is required for growth and cell division in bacteria [52; 57; 58]. Era homologs were found in _Drosophila_, mice, _Homo sapiens_[58], the plant _Antirrhinum majus_[59], the roundworm _Caenorhabditis elegans_[58], and in certain archaea [60]. Era depletion in eukaryotic cells led to G1 phase cell-cycle arrest and apoptosis [61]. In bacteria, Era deficiency results in increased levels of free 30S and 50S ribosomal subunits [62], accumulation of unprocessed 16S rRNA, and inhibition of translation [63]. The Era overexpression compensates for the _rbfA_ deletion that indicates Era participation in the maturation of the 30S subunit [27]. Era function probably overlaps RbfA. Era is a small two-domain protein (34 kDa in _E. coli_) (Figure 3c). The N-terminal GTP-binding domain of Era represents a six-stranded \(\beta\)-sheet surrounded by five \(\alpha\)-helices [64; 65]. It was shown that serine and threonine residues in the \({}_{33}\)ISITSR\({}_{38}\) sequence of the \(\beta\)2 strand of _E. coli_ Era could be phosphorylated [66]. The regulation of the Era activity via autophosphorylation is vital. Double substitution of Thr36 Ala and Ser37 Ala [66] and a point mutation of Ser34 Pro in _E. coli_ Era [67] were lethal. The C-terminal domain of Era consists of a three-stranded \(\beta\)-sheet and three \(\alpha\)-helices with \(\alpha\beta\alpha\alpha\beta\) topology, which is characteristic of the KH type II domain fold implicated in RNA binding [23]. Mutations in the GlyxxGly consensus of the KH domain of Era inhibited the Era's binding to the 16 SrRNA [68]. The two domains of Era are connected by a flexible linker, the length of which is important for ribosome biogenesis [63]. An insertion mutation with eight amino acid residues (AAANAAAAN) inserted into the Era's linker led to loss of suppression of the 30S maturation defects in the \(\Delta\)_rbfA_ strain. The cryo-EM structure of the mature 30S subunit in complex with Era demonstrated that the Era factor was located in the cleft between the head and platform, the opposite face of helix 28 from RbfA (Figure 4d) [69]. Era bound the anti-Shine-Dalgarno (anti-SD) \({}_{1531}\)AUACCUCUCUUA\({}_{1542}\) sequence at the 3\({}^{\prime}\)-end of the 16S rRNA. The \({}_{1531}\)AUCA\({}_{1534}\) region \"wraps around\" side chains of two lysine residues of the consensus \({}_{250}\)GlyLysLysGly\({}_{253}\) loop in the KH domain of Era [64]. Moreover, the cryo-EM structure of 30S subuniteEra complex revealed that binding of Era and ribosomal protein S1 was mutually exclusive [69]. In addition, Era may associate with the RNase YbeY and the RNA-helicase CshA [70]. Apparently, one of the roles of Era is to protect the anti-SD sequence from cleavage during the processing of the 3\({}^{\prime}\)-end of the 17S rRNA, and therefore block the duplex formation between the anti-SD of the 17S rRNA and the SD sequence of an mRNA, preventing premature translation. Cryo-EM analysis of immature 30S subunits obtained from Era-depleted _E. coli_ cells revealed that helices 23 and 24 of the platform failed to adopt the mature conformation [14]. In addition, helices 44 and 45 were disordered in the \(\Delta\)_era_ 30S particles, indicating that their maturation requires proper stabilization of helices 23 and 24. Interestingly, the 70S ribosome treatment with Era promoted ribosome dissociation and caused destabilization in h44 of the decoding center of the 30S subunit. The Era-induced destabilization of h44 inhibited YjeQ binding to the 30S subunit. Conversion of helix 44 to an immature state indicated that Era plays a role in ribosome homeostasis, including the reactivation of hibernating ribosomes or triggering of the ribosome degradation [14].