# Ribosomal Hibernation-Associated Factors in _Escherichia coli_
## 2 100S Ribosome  
Various types of ribosomal dimers have been observed using negative-staining electron microscopy since the 1970s [21; 22]. However, ribosomal dimers are not always observable by electron microscopy. Some of these complexes were thought to be linked by ribosome-bound mRNAs, whereas others were considered artificial because studies using sucrose density gradient centrifugation showed that ribosomes dimerized in the presence of excess divalent cations in solution. In the 1980s, [PERSON] et al. established an experimental method for stably obtaining ribosomal dimers [23] by avoiding the washing of _E. coli_ cells harvested during the stationary growth phase with fresh buffer. The stable ribosomal dimer, whose sedimentation coefficient was 100S, was analyzed using sucrose density gradient centrifugation and named the 100S ribosome. Subsequently, the protein RMF was found to bind to this ribosome, and it was confirmed that adding RMF to the ribosome in vitro caused dimerization (Figure 1) [12; 23]. The addition of RMF to ribosomes in vitro resulted in a marked decrease in translational activity, indicating that the 100S ribosome is inactive [12]. 100S ribosome formation is thought to inhibit the synthesis of proteins that consume large amounts of energy under conditions unfavorable for bacterial growth [8; 24]. According to the results of sucrose density gradient centrifugation, around 40% of all ribosomes are converted to 100S ribosomes during the stationary growth phase [23]. However, this measured value is uncertain because the 100S ribosome of _E. coli_ easily dissociates into 70S ribosomes at low ribosomal concentrations [25]. Cryo-electron microscopy tomography in situ revealed that 20-25% of ribosomes were in the 100S form; however, the culture conditions were not optimized to observe large numbers of these ribosomes [26]. In contrast, stoichiometric analysis of ribosomal binding proteins revealed that RMF binds to up to 80% or more of ribosomes in _E. coli_ cells [27]. Therefore, most ribosomes are thought to be converted to 100S ribosomes in the cell depending on the environment. In _E. coli_, ribosomes are degraded by exoribonucleases such as RNase R and RNase II in response to environmental stress [14; 15]. However, it has been speculated that the 100S ribosome complex protects ribosomes from degradation during osmotic or heat stress [28; 29]. Dormant cells known as persisters, which are resistant to antimicrobial drugs, contain high levels of 100S ribosomes [30]. When cells are removed from stress conditions, inactive 100S ribosomes quickly dissociate into active 70S ribosomes and protein synthesis resumes [31]. It has been reported that EF-G/RRF or Hflx is involved in the dissociation of 100S ribosome in _S. aureus_[32]. However, the 100S ribosome of _S. aureus_ is formed by the long form of HPF and not by RMF [10]. Although EF-G/RRF and Hflx are also present in _E. coli_, it has not been confirmed whether these protein factors can dissociate the 100S ribosome formed by RMF in _E. coli_. In _E. coli_, it has been reported that IF3 releases HPF from ribosomes but does not release RMF [33]. These studies suggest that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important strategy for cells to survive in harsh environments. This stage in the translational cycle, in which the ribosome has lost its activity,