Navratil Tomas, Spremulli Linda L
Department of Chemistry, Campus Box 3290, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Biochemistry. 2003 Nov 25;42(46):13587-95. doi: 10.1021/bi034855a.
Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to the A-site of the ribosome. In a multiple-sequence alignment of prokaryotic EF-Tu's, Gln97 is nearly 100% conserved. In contrast, in mammalian mitochondrial EF-Tu's, the corresponding position is occupied by a conserved proline residue. Gln97 is located in the switch II region in the GDP/GTP binding domain of EF-Tu. This domain undergoes a significant structural rearrangement upon GDP/GTP exchange. To investigate the role of Gln97 in bacterial EF-Tu, the E. coli EF-Tu variant Q97P was prepared. The Q97P variant displayed no activity in the incorporation of [(14)C]Phe on poly(U)-programmed E. coli ribosomes. The Q97P variant bound GDP more tightly than the wild-type EF-Tu with K(d) values of 7.5 and 12 nM, respectively. The intrinsic rate of GDP exchange was 2-3-fold lower for the Q97P variant than for wild-type EF-Tu in the absence of elongation factor Ts (EF-Ts). Addition of EF-Ts equalized the GDP exchange rate between the variant and wild-type EF-Tu. The variant bound GTP at 3-fold lower levels than the wild-type EF-Tu. Strikingly, the Q97P variant was completely inactive in ternary complex formation, accounting for its inability to function in polymerization. The structural basis of these observations is discussed.
延伸因子Tu（EF-Tu）将氨酰基tRNA转运至核糖体的A位点。在原核生物EF-Tu的多序列比对中，Gln97的保守率接近100%。相比之下，在哺乳动物线粒体EF-Tu中，相应位置被一个保守的脯氨酸残基占据。Gln97位于EF-Tu的GDP/GTP结合结构域的开关II区域。该结构域在GDP/GTP交换时会发生显著的结构重排。为了研究Gln97在细菌EF-Tu中的作用，制备了大肠杆菌EF-Tu变体Q97P。Q97P变体在以聚（U）编程的大肠杆菌核糖体上掺入[（14）C]苯丙氨酸时没有活性。Q97P变体比野生型EF-Tu更紧密地结合GDP，其解离常数（K(d)）值分别为7.5和12 nM。在没有延伸因子Ts（EF-Ts）的情况下，Q97P变体的GDP交换固有速率比野生型EF-Tu低2至3倍。添加EF-Ts使变体和野生型EF-Tu之间的GDP交换速率相等。该变体结合GTP的水平比野生型EF-Tu低3倍。引人注目的是，Q97P变体在三元复合物形成中完全无活性，这解释了其在聚合反应中无法发挥作用的原因。本文讨论了这些观察结果的结构基础。
