# Ribosomal Hibernation-Associated Factors in _Escherichia coli_
## 4 Factors Affecting 100S Ribosome Formation  
### Ribosome Modulation Factor  
RMF in _E. coli_ is a small, basic protein consisting of 55 amino acids whose main function is forming the 100S ribosome by binding to the 70S ribosome (see Table 1) (<12> in Figure 2). The _rmf_ gene is mainly carried by enteropathogenic bacteria belonging to the gammaprotebacteria family, such as _E. coli_, _Vibrio cholerae_, and _Yersinia pestis_[8]. Under culture conditions in a rich medium, RMF was not expressed during the exponential growth phase, but instead, its expression was initiated when the culture entered the stationary phase. The half-life of _rmf_ mRNA transcribed in the stationary phase is approximately 24 min, which is clearly longer than the typical half-life of _E. coli_ mRNA (2-4 min) [31], and its expression level is mainly regulated by ppGpp (<9> in Figure 2) [35]. Surprisingly, the expression of RMF is not dependent on RpoS (sigma S), the major alternative transcriptional factor in the stress and stationary phase in _E. coli_[35]. _rmf_ gene expression is reportedly regulated by numerous elements: cyclic AMP (cAMP) and cAMP receptor protein (CRP) (which are stimulated by a decrease in metabolic energy) (<10> in Figure 2); the transcription factor ArcA (which is involved in redox regulation under anoxic conditions); and the transcription factors McbR, RcdA, SdiA, and SlyA (which are involved in biofilm formation) (<11> in Figure 2) [52; 51]. Cells in a biofilm may develop antibiotic resistance by forming 100S ribosomes with reduced translational activity [53]. RMF is not only involved in the formation of ribosomal dimers, but also in the resistance of cells to osmotic stress [28], heat stress [29], and acid stress [54] by limiting rRNA damage [14; 15]. This indicates that RMF expression is controlled by a number of transcription factors that respond to various stresses [51].  
Figure 2: Ribosome cycle including the hibernation stage and expression pathways of factors related to formation of 100S ribosome in _Escherichia coli_. The state of ribosomes in the exponential phase, when cell proliferation is active, and in the stationary phase, when cells are under stress, are shown. The currently known expression pathways of RMF, HPF, and RaiA, factors involved in the formation of the 100S ribosome, are depicted.  
The binding sites of RMF on the ribosome have been investigated by protein-protein crosslinking [13], chemical probing [55], and structural analyses [18; 56]. The results of these structural analyses indicated that RMF binds near the anti-Shine-Dalgarno region of the 30S subunit (see Figure 1). The structure of the RMF consists of two helices connected by a linker, as determined in nuclear magnetic resonance analysis (PDB ID: 2 jRM), X-ray crystallography (PDB ID: 4V8G), and cryo-electron microscopy (PDB ID: 6H4N). Thirteen amino acids of RMF are completely conserved and functionally important, as replacement of one of these residues with alanine suppressed ribosome dimerization [57]. Examination of the functional sites using mutants of RMF revealed that R3, K5, and R11 in the N-terminal domain contribute significantly to ribosome binding. Analysis of the 100S ribosome by cryo-electron microscopy showed that R3 and K5 interact with helix 28 of 16S rRNA [18]. In addition, G23 and R45 are important for ribosome dimerization because although RMFs with mutations in these amino acids bind to ribosomes, they cannot form dimers [57]. Thus, RMF contains multiple functional sites for ribosome binding and ribosome dimerization.