# Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria
## 2 30S Ribosomal Subunit Biogenesis Factors
### Rim]  
A number of ribosomal proteins in _E. coli_ undergo post-translational modifications, but the functional significance of these modifications has remained unclear. Several enzymes perform post-translational modifications of ribosomal proteins. For example, enzymes Rim], RimI, and RimL acetylated the N-terminal residues of ribosomal proteins S5, S8, and L7/L12, respectively, and had structural similarities [88,89]. To date, it has been shown that RimJ is associated with the ribosome biogenesis [90]. RimJ is a single-domain protein (23 kDa in _Mycobacterium smegmatisis_) consisting of six \(\alpha\) helices and eight \(\beta\) strands with a NAT-domain fold typical for **N**-acetyltransferases (Figure 3e).  
The protein S5 takes part in the formation of the functionally important structure of 30S subunit, the central pseudoknot of the decoding center [9]. It was shown that the point mutation Gly28 Asp in the ribosomal protein S5 of _E. coli_ caused a cold-sensitive phenotype: growth was not detectable for this mutant strain at a reduced temperature (20 \({}^{\circ}\)C), which could have indicated defects in the ribosome biogenesis [90,91]. Indeed, the S5 (Gly28 Asp) mutant demonstrated increased amounts of free 30S and 50S subunits, accumulation of 17S rRNA, and reduced translation fidelity [90,91]. Deletion of _rimI_ exacerbated the growth defects of the S5 (Gly28 Asp) mutant. Still, overexpression of Rim] suppressed cold sensitivity and partially alleviated anomalous ribosome profiles and mRNA misreading associated with the S5 (Gly28 Asp) mutation [90]. Mass spectrometry analysis revealed that the S5 protein in both the wild-type and S5 (Gly28 Asp) 30S subunits was acetylated even without RimJ overexpression [90]. Furthermore, the elimination of acetyltransferase function by site-directed mutagenesis of two conserved cysteine residues at positions 54 and 105 in _E. coli_ Rim] did not disrupt the suppression activity of Rim] [90]. This RimJ mutant could still suppress the cold-sensitive phenotype of the S5 (Gly28 Asp) strain. Hence, this indicated that the acetyltransferase activeity is not required for the ability of Rim] to suppress the cold sensitivity caused by the Gly28 Asp change in S5. Moreover, deletion of the _rimI_ gene slowed growth, decreased the amounts of total protein per cell, and caused defects in ribosomal function [92]. So, Rim may have an additional function as a ribosome assembly factor, taking part in the remodeling of the structure of the central pseudoknot for stable association of protein S5 with its subsequent acetylation.  
Figure 5: A1518 and A1519 dimethylation is described in the main text. (**a**) Illustration of h44 and h45 (forest green) of the _E. coli_ 16S rRNA (PDB ID7 OE1). A1518 and A1519 nucleotides are shown in magenta. (**b**) Illustration of h45 (blue) and KsgA (green) extracted from the published structure of the \(\Delta\)kgsA pre-30S subuniteKsgA complex (PDB ID 705H). A1518 and A1519 are shown in orange.  
It has been shown that RimJ did not interact with mature 30S and 50S subunits, nor did it interact with 70S ribosomes [90]. Moreover, RimJ did not acetylate free protein S5, and only modified S5 in the context of the pre-30S subunit [32; 93]. It was found that the N-terminal acetylation of S5 occurred after the correct folding of the central pseudoknot of the pre-30S subunit [32; 94]. This suggested that this modification flagged the formation of specific RNA-protein contacts during assembly.