Tomsic Jerneja, Caserta Enrico, Pon Cynthia L, Gualerzi Claudio O
Laboratory of Genetics, Department of Bioscience and Biotechnology, University of Camerino, 62032 Camerino, Italy.
City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA.
Int J Mol Sci. 2021 Dec 8;22(24):13238. doi: 10.3390/ijms222413238.
Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.
翻译起始因子IF2的鸟苷酸结合结构域（IF2 G2）的三个共有元件之一中存在的保守组氨酸448被取代，导致核糖体依赖性GTP酶活性受损，阻止了IF2从核糖体上解离，引起严重的蛋白质合成抑制，并产生显性致死表型。先前已表明，IF2对核糖体的亲和力降低可抑制这种致死性。在此，我们证明，IF2对fMet-tRNA的亲和力降低也可抑制这种显性致死表型，并允许IF2在完全不存在GTP水解的情况下支持准确翻译。这些结果强化了这样一个前提，即翻译起始后期核糖体、IF2和fMet-tRNA发生的构象变化是由热驱动的，并且IF2依赖性GTP水解产生的能量对于成功的翻译起始不是必需的，并且IF2 C2与fMet-tRNA受体末端之间的相互作用解离，这是在形成具有延伸能力的70S复合物之前将该因子锚定到核糖体的最后一个纽带，对于fMet-tRNA在有效P位点的调整和IF2从核糖体上的释放都是限速的。
