Jagannathan Indu, Culver Gloria M
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 4258 Molecular Biology Building, Ames, IA 50011, USA.
J Mol Biol. 2003 Jul 4;330(2):373-83. doi: 10.1016/s0022-2836(03)00586-2.
Assembly of the 30S 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 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S 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 30S 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 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S 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 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.
30S核糖体亚基的组装以高度有序且连续的方式进行。核糖体蛋白按顺序添加到不断增长的核糖核蛋白颗粒上，这一过程由主要结合蛋白的结合引发。这些蛋白特异性且独立地结合到16S核糖体RNA（rRNA）上。两种主要结合蛋白S8和S15仅与16S rRNA的中央结构域相互作用。S15与中央结构域的结合导致RNA构象发生变化，随后S6/S18二聚体、S11以及最后S21有序组装，形成30S亚基的平台。相比之下，S8不是这个主要平台组装分支的一部分。在其余与中央结构域结合的蛋白中，只有S21的结合略微依赖于S8。因此，尽管S8是一种与中央结构域广泛接触的主要结合蛋白，但其在该结构域组装中的作用仍不清楚。在这里，我们从S15上的四个独特位置进行定向羟基自由基探测，以评估由于S8结合而导致的16S rRNA中央结构域的组织情况。对Fe(II)-S15/16S rRNA和Fe(II)-S15/S8/16S rRNA核糖核蛋白颗粒进行羟基自由基探测，结果显示添加S8后，S15的16S rRNA环境发生了变化。这些变化主要发生在先前确定的S8结合位点附近的螺旋24和螺旋26中。这些依赖于S8的构象变化与完整30S亚基中16S rRNA的折叠情况一致。因此，虽然平台的组装并非绝对需要S8的结合，但它似乎通过影响中央结构域的组织，显著影响了S15的16S rRNA环境。
