Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Biochemistry. 2011 Feb 8;50(5):854-63. doi: 10.1021/bi101005r. Epub 2011 Jan 11.
KsgA is an rRNA methyltransferase important to the process of small subunit biogenesis in bacteria. It is ubiquitously found in all life including archaea and eukarya, where the enzyme is referred to as Dim1. Despite the emergence of considerable data addressing KsgA function over the last several years, details pertaining to RNA recognition are limited, in part because the most accessible substrate for in vitro studies of KsgA is the 900000 Da 30S ribosomal subunit. To overcome challenges imposed by size and complexity, we adapted recently reported techniques to construct in vivo assembled mutant 30S subunits suitable for use in in vitro methyltransferase assays. Using this approach, numerous 16S rRNA mutants were constructed and tested. Our observations indicate that the 790 loop of helix 24 plays an important role in overall catalysis by KsgA. Moreover, the length of helix 45 also is important to catalysis. In both cases loss of catalytic function occurred without an increase in the production of N(6)-methyladenosine, a likely indication that there was no critical reduction in binding strength. Both sets of observations support a "proximity" mechanism of KsgA function. We also report that several of the mutants constructed failed to assemble properly into 30S subunits, while some others did so with reduced efficiency. Therefore, the same technique of generating mutant 30S subunits can be used to study ribosome biogenesis on the whole.
KsgA 是一种 rRNA 甲基转移酶，对细菌小亚基生物发生过程至关重要。它普遍存在于所有生命形式中，包括古菌和真核生物，在这些生物中，该酶被称为 Dim1。尽管在过去几年中已经有相当多的关于 KsgA 功能的研究数据，但关于 RNA 识别的细节是有限的，部分原因是最容易获得的用于 KsgA 体外研究的底物是 900000 Da 的 30S 核糖体亚基。为了克服大小和复杂性带来的挑战，我们采用了最近报道的技术来构建适合用于体外甲基转移酶测定的体内组装突变 30S 亚基。使用这种方法，构建并测试了许多 16S rRNA 突变体。我们的观察表明，螺旋 24 的 790 环在 KsgA 的整体催化中起着重要作用。此外，45 号螺旋的长度对催化也很重要。在这两种情况下，催化功能的丧失都没有导致 N(6)-甲基腺苷的产生增加，这很可能表明结合强度没有明显降低。这两组观察结果都支持 KsgA 功能的“接近”机制。我们还报告说，构建的几个突变体不能正确组装到 30S 亚基中，而有些突变体则以较低的效率组装。因此，生成突变 30S 亚基的相同技术也可用于研究整个核糖体生物发生。
