# Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria
## 2 30S Ribosomal Subunit Biogenesis Factors  
### RbfA  
Ribosome-binding factor\(\underline{\mathbf{\Delta}}\) (RbfA) is a small single-domain protein (13-15 kDa) consisting of three \(\alpha\) helices and three \(\beta\) strands with a type-II K homology (KH)-domain fold topology (Figure 3a) [22]. The KH domain has a characteristic helix-turn-helix consensus GxxG motif involved in the RNA recognition [23; 24]. Following the GxxG binding motif of the KH domain, RbfA exhibits a preference for the AxG sequence [22]. RbfA is found in the majority of bacteria and archaea, and RbfA orthologs occur in eukaryotic mitochondria and chloroplasts [25; 26]. Deletion of the _rbfA_ gene results in slow-growing bacteria and the  
Figure 2: Surface of the 30S subunit of _Escherichia coli_ (PDB ID: 70E1) (inter-subunit interface view). Distinct structural domains of the 30S subunit (the head, the body, and the platform) are shown in light wheat, pale white, and light pink, respectively. The helices of the decoding center (CDR)—h1, h2, h27, h28, h44, and h45—are shown as illustrated models. accumulation of free ribosomal subunits and unprocessed 16S rRNA (17S rRNA) [12; 27]. RbfA was initially found as a multicopy suppressor of the cold sensitivity C23U mutation of 16S rRNA, locating at the 5\({}^{\prime}\) terminal helix (h1) [28]. RbfA overcomes the translational block at low temperature, promoting the correct folding and maturation of the 5\({}^{\prime}\)-end of the 16S rRNA [29]. Figure 3: Domain organization of the 30S assembly factors. Secondary structure elements, \(\alpha\)-helices (\(\alpha\)), \(\beta\)-strands (\(\beta\)), and 3\({}_{10}\) helices (\(\alpha^{\prime}\)) are numbered according to their relative position in the polypeptide sequence. Crystal structures of: (**a**) RbfA (PDB ID: 1 KKG); (**b**) YjeQ (RsqA) (PDB ID: 1 UOL); (**c**) Era (PDB ID: 1 EGA); (**d**) KsqA (RsmA) (PDB ID: 1 QYR); (**e**) RimJ (PDB ID: 6C30); (**f**) RimM (PDB ID: 2 DYI); (**g**) RimP (PDB ID: 7 NAT); (**h**) Hfq (PDB ID: 1 HK9). Two cryo-EM structures of the 30S subunit in complex with RbfA did not reveal any direct interactions between RbfA and helix 1 of 16S rRNA [29; 30]. Moreover, these structures showed different RbfA localization on the small ribosomal subunit (Figure 4a,b). RbfA binds in the neck region (helix 28), being buried within the cleft between the head and the body of the 30S subunit [29; 30]. In one structure, RbfA is localized at the interface of the 30S subunit: the C-terminus of RbfA extends toward the 5\({}^{\prime}\)-end of the 16S rRNA, and the N-terminal RNA-binding interhelical kink with an AxG motif (helix \(\alpha\)2-turn-helix \(\alpha\)3) faces the junction between h44 and h45, in that the top of helix 44 is displaced [29]. In another structure, the RNA-binding AxG motif of RbfA interacts with the 3\({}^{\prime}\)-end of the 16S rRNA [30]. Interestingly, for RbfA binding, the 3\({}^{\prime}\)-end of the 16S rRNA must be in the exit channel. The rearrangement of the 3\({}^{\prime}\)-end of the 16S rRNA from the entry to the exit of the mRNA channel is accompanied by reorganization of the helix 28 [9]. It has been suggested that RbfA promotes the conversion of h28 to a mature state, and holds the 3\({}^{\prime}\)-end of the 16S rRNA at the exit of the mRNA channel [30]. The upper part of h28 is displaced, and the lower end of h28 is destabilized, thus keeping h28 from adopting a fully matured conformation [30]. Nucleotides of the disordered lower part of the helix 28 play an important role in stabilizing the h44/45 linker in the mature 30S subunit. Therefore, RbfA delay the folding of the h44/45 linker and promote the correct folding of adjacent h1 via h28. It was shown that RbfA acts as a \"gatekeeper\" to prevent premature entry of pre-30S subunits into the translation cycle. RbfA inhibits protein synthesis by pre-30S subunits in the presence of translation initiation factor IF3 [31]. IF3 was able to release RbfA from mature 30S subunits, but not from immature 30S particles.