# Ribosomal Hibernation-Associated Factors in _Escherichia coli_
## 5 Conclusions  
The ribosome cycle in bacteria consists of initiation, elongation, termination, and recycling stages, as shown in Figure 2. During the exponential growth phase, the ribosome progresses step-by-step in this cycle (<1> \(\rightarrow\) <2> \(\rightarrow\) <3> \(\rightarrow\) <4> \(\rightarrow\) <5> \(\rightarrow\)<1> in Figure 2), during which proteins are synthesized. However, the protein synthesis activity in bacteria is inhibited under stressful conditions, such as amino acid starvation. Upon amino acid starvation, the number of deacylated tRNAs initially increases; these tRNAs bind to the A site of the ribosome (<6> in Figure 2). Once bound, RelA binds to the ribosome and initiates the synthesis of (p)ppGpp (<7> in Figure 2). Under stress conditions, such as fatty acid starvation, carbon source starvation, phosphate starvation, and hyperosmotic shock, SpoT synthesizes (p)ppGpp (<8> in Figure 2). Transcription of the _rmf_ gene is mainly induced by (p)ppGpp, which is synthesized by RelA and/or SpoT (<9> in Figure 2) and regulated by the cAMP-activated global transcriptional regulator CRP (<10> in Figure 2) and the transcription factors involved in biofilm formation McbR, RcdA, SdiA, and SlyA (<11> in Figure 2). RMF, translated as a protein factor, competes with IF3 for binding to ribosomes that have completed the recycling step (determining whether the ribosome proceeds to <5> or <12> in Figure 2). Prior to this event, as the _E. coli_ culture transitions from the exponential phase to the stationary phase, changes to the ribosome make it less likely to bind to IF3 and more likely to form the 100S ribosome mediated by RMF. As ribosomal proteins, S1 and IF3 are bound to the 30S subunit in the initiation stage and inactive S1 and RMF are bound to the 100S ribosome, S1 protein may be involved in the ribosomal changes described above. The binding of RMF results in the formation of a 70S dimer, which is recognized as a particle with a sedimentation coefficient of 90S in sucrose density gradient centrifugation (<13> in Figure 2). The binding of HPF to the unstable 90S particle transforms it into a mature 100S ribosome (<14> in Figure 2). HPF expression is known to be controlled by autoinducer 2. In contrast, RaiA, a paralog of HPF, suppresses the formation of the 100S ribosome by binding to the 70S ribosome (<15> in Figure 2). Its expression is under the control of (p)ppGpp (<18> in Figure 2) and cAMP-CRP (<17> in Figure 2) as well as RMF. Some of these 70S and 100S ribosomes, which have lost their translation activity during the stationary phase, are localized near the membrane by YqjD (<19> in Figure 2). When the surrounding environment of the cell is improved, the 100S ribosome is immediately converted to an active 70S ribosome by releasing RMF and HPF, and the ribosome cycle resumes (<20> in Figure 2). This ribosomal hibernation system is thought to be suitable for the life cycle of enterobacteria, which are released from starvation and multiply at once when their host eats. The genes for these ribosomal hibernation factors are scattered throughout the _E. coli_ genome (see Table 1) and use different expression pathways (see Figure 2). The expression levels of these proteins may be regulated according to the type of stress. The formation of 100S ribosomes is a key strategy for bacterial survival under various stress conditions, which protects bacteria in the presence of antibiotics. Therefore, determining the expression mechanism of protein factors, such as RMF and HPF, which contribute to the formation of 100S ribosomes, will be useful for considering the effective use of antibiotics. [PERSON] wrote sections on the 100S ribosome and RMF; [PERSON] wrote the section on RaiA and HPF. All authors have read and agreed to the published version of the manuscript.