Fischer Niels, Paleskava Alena, Gromadski Kirill B, Konevega Andrey L, Wahl Markus C, Stark Holger, Rodnina Marina V
3D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
Biol Chem. 2007 Oct;388(10):1061-7. doi: 10.1515/BC.2007.108.
In bacteria, UGA stop codons can be recoded to direct the incorporation of selenocysteine into proteins on the ribosome. Recoding requires a selenocysteine incorporation sequence (SECIS) downstream of the UGA codon, a specialized translation factor SelB, and the non-canonical Sec-tRNASec, which is formed from Ser-tRNASec by selenocysteine synthase, SelA, using selenophosphate as selenium donor. Here we describe a rapid-kinetics approach to study the mechanism of selenocysteine insertion into proteins on the ribosome. Labeling of SelB, Sec-tRNASec and other components of the translational machinery allows direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer between two fluorophores. Furthermore, the structure of SelA was studied by electron cryomicroscopy (cryo-EM). We report that intact SelA from the thermophilic bacterium Moorella thermoacetica (mthSelA) can be vitrified for cryo-EM using a controlled-environment vitrification system. Two-dimensional image analysis of vitrified mthSelA images shows that SelA can adopt the wide range of orientations required for high-resolution structure determination by cryo-EM. The results indicate that mthSelA forms a homodecamer that has a ring-like structure with five bilobed wings, similar to the structure of the E. coli complex determined previously.
在细菌中，UGA终止密码子可被重新编码，以指导硒代半胱氨酸在核糖体上掺入蛋白质。重新编码需要UGA密码子下游的硒代半胱氨酸掺入序列（SECIS）、一种特殊的翻译因子SelB以及非经典的Sec-tRNASec，后者由丝氨酸-tRNASec通过硒代半胱氨酸合酶SelA利用硒代磷酸酯作为硒供体形成。在此，我们描述了一种快速动力学方法，用于研究硒代半胱氨酸在核糖体上插入蛋白质的机制。对SelB、Sec-tRNASec和翻译机制的其他组分进行标记，通过监测单个染料荧光的变化或两个荧光团之间的荧光共振能量转移，可直接观察复合物的形成或解离。此外，还通过冷冻电子显微镜（cryo-EM）研究了SelA的结构。我们报告称，嗜热细菌嗜热栖热放线菌（mthSelA）的完整SelA可使用可控环境玻璃化系统进行玻璃化以用于冷冻电镜分析。对玻璃化的mthSelA图像进行二维图像分析表明，SelA可呈现冷冻电镜进行高分辨率结构测定所需的广泛取向。结果表明，mthSelA形成一种同十聚体，其具有类似先前确定的大肠杆菌复合物结构的带有五个双叶状翼的环状结构。
