# Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria
## 2 30S Ribosomal Subunit Biogenesis Factors
### KsgA (RsmA)  
KsgA (also known as RsmA\(\bullet\)Ribosomal RNA small subunit methyltransferase A) was identified upon selecting _E. coli_ strains resistant to the aminoglycosable antibiotic kasugamycin due to the lack of A1518 and A1519 dimethylation of the 16S rRNA [73,74]. KsgA is (adenine-N6, N6-)-dimethyltransferase, which dimethylates the two adjacent adenosines of the helix 45, A1518 and A1519 [73]. Lack of A1518 and A1519 dimethylation caused severe damage to the translation [75; 76]. A1518 and A1519 are universally conserved residues, and KsgA occurs in all three domains of life [77; 78], suggesting that the modification of the two adjacent adenosine residues has an important role in cells. Nevertheless, _ksgA_ was not required for the survival of _E. coli_[79], whereas its knockout was lethal in yeasts [80]. It was shown that deletion of _ksgA_ substantially slowed cell growth of _E. coli_[81]. This effect was even more substantial at lower temperatures. The _alsgA_ cells accumulated free ribosomal subunits and 17S rRNA. Surprisingly, the overexpression of a methyltransferase-inactive form of KsgA (Glu66 Ala) in the _alsgA_ strain led to a dramatically reduced growth rate at all temperatures, as well as accumulation up to 95% of free 30S subunits containing the 17S rRNA [81]. It turned out that catalytically inactive KsgA strongly associated with the pre-30S subunits and stalled the 17S rRNA processing, excluding premature subunits from the translation cycle. These data indicated that KsgA's role goes beyond the methylation of the 16S rRNA, and KsgA is integrated into the network of other 30S assembly factors. So, methylation is a second function for KsgA, while its primary and critical role is monitoring the 30S subunit biogenesis. It was shown that KsgA had a low affinity for methylated 30S subunits [74]. The methylation was assumed to be a trigger for release of KsgA from assembling 30S subunits, and the release of KsgA was accompanied by conformational rearrangements that allowed final maturation and entrance into the translation cycle. KsgA consists of two domains with 30 kDa molecular mass (for _E. coli_) (Figure 3d). The N-terminal catalytical domain of KsgA has a Rossmann-like fold and three additional 3\({}_{10}\)-helices [78]. This Rossmann-like fold consists of a central seven-stranded \(\beta\)-sheet sandwiched between three \(\alpha\)-helices on one side and four on the other. The C-terminal domain of KsgA comprises four \(\alpha\)-helices and one 3\({}_{10}\)-helix. It was shown that KsgA was unable to methylate 30S subunits in the translationally active conformation. At the same time, it could modify 30S subunits when they were in an experimentally well-established, translationally inactive conformation, indicating that KsgA recognized a specific conformational state of the 30S ribosomal subunit [82]. It was found to require the presence of a minimal set of ribosomal proteins for methylation: proteins of platform S6, S11, S15, S18; and body S4, S8, S16, S17 [82; 83]. The structures of the 30S ribosomal subunit with KsgA have been determined for mature 30S subunit incubated with KsgA after depletion of magnesium ions [30; 84], and for the immature 30S subunit from the _alsgA_ cells also under low Mg\({}^{2+}\) concentration conditions [85]. The mature 30S subunits could hold KsgA, as they mimicked the methylation-ready conformation under the low-magnesium conditions [84]. Cryo-EM data for the 30S subunit\(\bullet\)KsgA complex were consistent with the results of hydroxyl-radical footprinting, according to which KsgA bound to the 30S subunit at the inter-subunit interface around the decoding center and the platform (Figure 4e) [86]. The C-terminal domain of KsgA bound to helices 27 and 24 of the 16S rRNA (platform), and the N-terminal domain of KsgA contacted the tetraloop of the helix 45 where A1518 and A1519 were located [30; 84; 85]. KsgA contacted the h45 so that the h44 could not access its native position [16; 81].