Ilag Leopold L, Videler Hortense, McKay Adam R, Sobott Frank, Fucini Paola, Nierhaus Knud H, Robinson Carol V
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8192-7. doi: 10.1073/pnas.0502193102. Epub 2005 May 27.
Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these high-resolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7/L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.
核糖体是将遗传密码普遍翻译为蛋白质的分子，代表着不对称、通常异质且包含动态区域的大分子结构。这些特性给现代结构生物学带来了相当大的挑战。尽管存在这些障碍，但嗜热栖热菌、耐辐射球菌和死海嗜盐菌核糖体的30S和50S亚基的高分辨率X射线结构已经揭示了RNA结构及其与蛋白质的相互作用。然而，由于其高构象动力学和潜在的异质性，一些区域仍然无法通过这些高分辨率方法获得，特别是所谓的L7/L12柄复合物。该区域通过在翻译过程中与GTPase因子相互作用，在蛋白质合成中发挥着至关重要的作用。在这里，我们应用串联质谱，这是一种广泛应用于蛋白质组学肽测序但以前未应用于MDa复合物的方法。对完整30S和50S亚基分配的离子进行分离和激活，分别释放出蛋白质S6和L12。重要的是，这个过程专门揭示了附着在核糖体上时L12的磷酸化，L12是位于柄复合物顶端多个拷贝中的蛋白质。此外，通过串联质谱，我们发现了嗜热栖热菌和其他嗜热菌上柄突出物的化学计量，并将这种组装与嗜温菌核糖体上的类似组装进行了对比。连同与降解体潜在相互作用的证据，这些结果表明，即使是对研究充分的嗜热栖热菌核糖体，串联质谱也能发现关于核糖体结构、相互作用和修饰的重要发现。
