Assembly of the Central Domain of the 30 S Ribosomal Subunit: Roles for the Primary Binding Ribosomal Proteins S15 and S8

Assembly of the 30 S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16 S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16 S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30 S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16 S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)–S15/16 S rRNA and Fe(II)–S15/S8/16 S rRNA ribonucleoprotein particles reveal changes in the 16 S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16 S rRNA folding in complete 30 S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16 S rRNA environment of S15 by influencing central domain organization.