Marzi Stefano, Knight William, Brandi Letizia, Caserta Enrico, Soboleva Natalia, Hill Walter E, Gualerzi Claudio O, Lodmell J Stephen
Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC) Italy.
RNA. 2003 Aug;9(8):958-69. doi: 10.1261/rna.2116303.
Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.
细菌翻译起始因子IF2是一种GTP结合蛋白，可催化起始甲硫氨酰-tRNA在核糖体P位点的结合。IF2在核糖体亚基上的拓扑定位是理解起始复合物形成机制的前提条件，但一直难以捉摸。在此，我们提出了一个IF2在70S起始复合物中定位的模型，该模型是通过将化学核酸酶Cu(II):1,10-邻菲罗啉和Fe(II):EDTA与引入IF2的半胱氨酸残基相连，对rRNA进行切割来确定的。IF2的GII结构域中的两个特定氨基酸靠近16S rRNA的H3、H4、H17和H18螺旋。此外，C-1和C-2结构域的交界处靠近23S rRNA的H89和硫链丝菌素区域。C-2结构域与P位点结合的tRNA的必要接近以及IF2保守的GI结构域与大亚基的因子结合中心，进一步限制了对接。将我们目前的发现与先前的数据进行比较，进一步表明在从30S到70S起始复合物的转变过程中，IF2在30S亚基上的方向发生了变化。
