# Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria
## 2 30S Ribosomal Subunit Biogenesis Factors
### Network of 30S Assembly Factors  
Here, we systematized the biochemical and genetic data on the described above ribosome biogenesis factors to build up a network of functional interactions between the 30S assembly factors (Figure 6). In this scheme, the arrows connecting two factors indicate negative (\(-\)), positive (\(+\)), or lack (\(\times\)) of the effect on the ribosome biogenesis in a strain deleted for one factor (located at the arrowhead) by an excess of another factor provided in trans (located at the tail). Positive relationships (synergy or additivity) indicate that functions of the two factors either overlap or are related to each other; whereas in the case of negative relationships, factors act as antagonists. Such relationships imply both functional redundancy and certain hierarchy for some of the ribosome biogenesis factors. This network, along with structural analysis of assembly intermediates, reveal a temporalframework and an order of action of these factors during the maturation of the CDR. Therefore, we described the time course of the events during the CDR maturation assisted by the selected 30S assembly factors. We propose the following sequence of the factor actions: RimM + Era > RbfA > RimP > RimJ > YjeQ > KsgA > Hfq. Factors RimM, Era, RbfA, RimP, and RimJ were assigned to a group of the \"early\"-stage factors that take part in the formation of the central pseudoknot, including folding of the head. Some \"early\"-stage factors assist the CDR maturation throughout the entire process; for example, RimP and Era. The remaining factors (YjeQ, KsgA, and Hfq) belong to a group of \"late\"-stage factors, which are required for positioning of the helix 44 and processing of the 5\({}^{\prime}\)- and 3\({}^{\prime}\)-ends of the 16S rRNA. Ribosome biogenesis occurs in the 5\({}^{\prime}\)-to-3\({}^{\prime}\) direction. We suggest that the \"early\"-stage factors of RimM and Era are among the first to take part in the 30S subunit assembly. It appears that RimM binds the 3\({}^{\prime}\) major domain cotranscriptionally, together with protein S19, and facilitates the assembly of the head. At the same time, Era interacts with the 3\({}^{\prime}\)-end of the 17S rRNA during the excision of the 16S rRNA precursor from the primary transcript and protects the anti-SD sequence from cleavage. The early association of Era with the 3\({}^{\prime}\)-end of the 17S rRNA and the Era-promoted platform stabilization may induce the movement of the 3\({}^{\prime}\)-end to the exit of the maturing mRNA channel. In turn, it facilitates the RbfA binding to the pre-30S subunits. As a gatekeeper, RbfA is bound within the mRNA channel exit, where it captures the 3\({}^{\prime}\)-end region of the 17S rRNA upstream of the Era-binding site. Therefore, RbfA blocks the mRNA interaction with the pre-30S subunit, preventing premature translation, as does Era. The positioning of the 3\({}^{\prime}\)-end of the 17S rRNA at the exit of the mRNA channel is accompanied by conversion of h28 into the mature form. This conversion is assisted by RbfA. The assembly factor RbfA distorts the upper region and destabilizes the lower region of h28. These conformational changes in the helix 28 impede folding of the h44/45 linker. Apparently, the delay in the folding of the h44/45 linker is required for the formation of central pseudoknot (PK) helices 1 and 2. The accurate formation and stability of the central PK is a critical step during the small subunit assembly. The central PK connects the head, body, and platform with each other. During the early stages of the 30S subunit biogenesis, premature central PK formation is blocked by a structure within the unprocessed 5\({}^{\prime}\)-region (the 5\({}^{\prime}\)-leader sequence) of the 17S rRNA that is mutually exclusive with h1. During the CDR folding, RbfA is implicated in the restructuring of the 5\({}^{\prime}\)-leader sequence of the 17S  
Figure 6: Network of functional interactions between the 30S assembly factors.