Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits:: A functional role for the N-terminus of the factor
Binding of Escherichia coli initiation factor IF2 to 30S ribosomal subunits:: A functional role for the N-terminus of the factor
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DOI：
10.1006/bbrc.1998.9664
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发表时间：
1998-11-18
影响因子：
3.1
通讯作者：
Sperling-Petersen, HU
中科院分区：
文献类型：
--
作者：
Moreno, JMP;Kildsgaard, J;Sperling-Petersen, HU
在细菌蛋白质合成的起始步骤中，启动因子IF2必须加入30S核糖体亚基，以促进tRNA(F)(Met)的结合。为了确定IF2中可能参与初级核糖体-因子相互作用的区域，我们构建了该因子的几个C端和N端截短形式以及分离的结构域，并在体外进行了30S核糖体结合试验。在这些实验中使用了表位位于IF2的两个N-末端结构域的单抗。到目前为止，还没有功能被分配给IF2的N端子区域。在这里，我们证明了由两个N-末端结构域组成的突变体与核糖体亚基具有内在的亲和力。此外，缺少两个N-末端结构域的IF2缺失突变体的亲和力可以忽略不计。此外，表位位于结构域II的单抗强烈抑制IF2与30S核糖体亚基的结合能力，而表位位于结构域I的单抗不影响因子的结合。IF2的C末端结构域与核糖体小亚基没有亲和力。此外，具有C末端缺失的突变体在这种相互作用中不会受到显著影响。因此，我们得出结论，IF2的N末端本身具有与核糖体亚基结合的亲和力，结构域II直接参与了相互作用。(C)1998年学术出版社。
In the initiation step of bacterial protein synthesis initiation factor IF2 has to join the 30S ribosomal subunit in order to promote the binding of the tRNA(f)(Met). In order to identify regions within IF2 which may be involved in the primary ribosome-factor interaction, we have constructed several C-terminal and N-terminal truncated forms of the factor as well as isolated structural domains, and tested them in a 30S ribosomal binding assay in vitro. Monoclonal antibodies with epitopes located within the two N-terminal domains of IF2 were used in these experiments. Hitherto, no function has been allocated to the N-terminal region of IF2. Here we show that a mutant consisting of the two N-terminal domains has intrinsic affinity to the ribosomal subunit. Furthermore, a deletion mutant of IF2 which is lacking the two N-terminal domains shows negligible affinity. Moreover mAb with epitopes located within domain II strongly inhibits the binding capacity of IF2 to the 30S ribosomal subunit, whereas mAb with epitopes mapped within domain I do not affect the binding of the factor. The C-terminal domain of IF2 shows no affinity for the small ribosomal subunit. In addition, mutants with C-terminal deletions are not significantly affected in this interaction. Therefore, we conclude that the N-terminus of IF2 has affinity per se to bind the ribosomal subunit, with domain II being directly involved in the interaction. (C) 1998 Academic Press.