# Ribosomal Hibernation-Associated Factors in _Escherichia coli_
## 4 Factors Affecting 100S Ribosome Formation
### RaiA and HPF  
Protein Y, the gene product of _yfiA_, was identified as a factor binding to the intersubunit position of the 30S, which stabilizes the 70S monomer of _E. coli_ ribosomes under low Mg\({}^{2+}\) conditions in vitro [58]. Subsequently, this protein was found to inhibit cell-free translation of mRNA in cell extracts of _E. coli_ and be induced under cold-shock stress. Thus, protein Y was renamed as ribosome-associated inhibitor A (RaiA) and its gene name (_yfiA_) as _raiA_, as shown in Table 1[59]. It has also been reported that RaiA reduces translation errors [60].  
RaiA and its paralogous protein YhbH were found to be associated with hibernating ribosomes in the stationary phase of _E. coli_ cells and were released from translating ribosomes soon after starved cells were transferred into a fresh medium. This indicates that these proteins are involved in the storage form of ribosomes in the stationary phase, at least in _E. coli_, as is the case with RMF [61]. YhbH appeared to promote the formation of 100S ribosomes by converting the immature 90S dimer containing RMF; hence, the protein and its gene name were renamed as HPF and _hpf_, respectively, as shown in Table 1 (<14> in Figure 2) [62]. Although both RaiA and HPF are encoded in the _E. coli_ genome and are highly homologous to each other, HPF binds to 100S ribosomes with RMF in contrast to RaiA binding to the 70S monomer in the ribosomal fraction (<15> in Figure 2) [61].  
Crystallographic and chemical probing analyses revealed that RaiA blocks the peptidyltRNA site (P site) and aminoacyl-tRNA site (A site) of the ribosome and inhibits translation initiation [63]. It was suggested that the HPF binding site overlaps with RaiA because of the high identity of these sequences and their similar structures in solution [64]; nevertheless, HPF promotes 100S formation with RMF, in contrast to RaiA, which prevents dimer formation [62]. The 3D structure of HPF and YfiA on the ribosome showed that these proteins are bound in the channel between the head and body of the 30S subunit where tRNAs and mRNA bind during protein synthesis [56]. It was also suggested that RaiA inhibits 100S formation using its C-terminal short tail, which blocks binding of RMF and prevents RMF-induced dimer formation. Recently, a high-resolution structure of hibernating 100S ribosomes from _E. coli_ was reported with well-resolved electron densities for HPF and RMF, revealing a direct interaction of HPF with E-site tRNA, as shown in Figure 1[18].  
Some information on the expression and regulatory mechanisms of the genes of RaiA and HPF in _E. coli_ is known. Expression of both proteins is induced under starved conditions [61], including nitrogen starvation [65]. It has also been reported that the mRNA abundance of _hpf_ is increased by the signal of autoinducer 2 (<16> in Figure 2) [66]. RaiA was induced under cold-shock treatment [59] and downregulated by FNR, a global regulator of anaerobic metabolism [67]. RaiA and RMF expression is induced by cAMP in response to glucose starvation (<17>, <10> in Figure 2) [51] and by starvation alarmome (p)ppGpp (<18>, <9> in Figure 2) [68].  
Recently, some results concerning functions of these proteins and storage ribosomes have been reported in several bacterial species. The HPF- or RaiA-bound storage ribosomes of _E. coli_ exhibit resistance to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases, with the intrinsic chaperon activity retained to assist in protein folding [69]. The absence of HPF results in the loss of some proteins from the ribosome during incubation in the stationary phase [70; 71]; furthermore, HPF protects ribosomes against degradation in the absence of mRNA by blocking the attack of ribonuclease [72; 73; 74]. Although these results include cases of specific species except for gammaproteobacteria, the inter-subunit proteins of non-active ribosomes may typically protect the machinery from degradation under stress conditions.