La Teana A, Gualerzi C O, Brimacombe R
Department of Biology, University of Camerino, Italy.
RNA. 1995 Oct;1(8):772-82.
The hypothesis of an adjustment of the mRNA in its ribosomal channel under the influence of the initiation factors has been tested by site-directed crosslinking experiments. Complexes containing 30S subunits with bound mRNA having 4-thio-uracil at specific positions were prepared in the presence or absence of initiation factors and/or fMet-tRNA and subjected to UV irradiation to obtain specific crosslinks of the radioactively labeled mRNA with bases of the 16S rRNA and with ribosomal proteins. The subsequent identification of the specific sites of both mRNA and rRNA and individual ribosomal proteins involved in the crosslinking, obtained under different conditions of complex formation, provide direct evidence for the occurrence of a partial relocation of the mRNA on the 30S ribosomal subunits under the influence of the factors. The nature of this mRNA relocation is compatible with our previous proposal of a shift of the template from an initial ribosomal "stand-by site" to a second site closer to that occupied when the initiation triplet of the mRNA is decoded in the P-site.
通过定点交联实验对起始因子影响下mRNA在核糖体通道中发生调整的假说进行了验证。在有或没有起始因子和/或fMet - tRNA的情况下，制备含有30S亚基与在特定位置带有4 - 硫尿嘧啶的结合mRNA的复合物，并进行紫外线照射，以使放射性标记的mRNA与16S rRNA的碱基以及核糖体蛋白形成特异性交联。随后对在不同复合物形成条件下获得的参与交联的mRNA、rRNA和单个核糖体蛋白的特定位点进行鉴定，为在这些因子影响下mRNA在30S核糖体亚基上发生部分重新定位提供了直接证据。这种mRNA重新定位的性质与我们之前提出的模板从初始核糖体“备用位点”转移到更接近mRNA起始三联体在P位点解码时所占据位点的第二个位点的提议相符。
