Graĭfer D M, Zenkova M A, Malygin A A, Matasova N B, Mundus D A, Karpova G G
Mol Biol (Mosk). 1995 Jan-Feb;29(1):114-24.
Region 980-1061 in human 18S rRNA was chosen on the basis of our previous results indicating, that the cross-linking sites of alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their ability to bind selectively at the desired rRNA sequences was proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Only four of the probes were able to bind to 40S subunits indicating, that the corresponding 18S rRNA sequences 980-994, 987-1001, 1025-1039 and 1032-1046 are exposed within the subunits. None of the probes inhibited tRNA-dependent binding of oligo(U) messengers to 40S subunits. Nevertheless, two probes (complementary to 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA.eIF-2.GTP was strongly affected by the same oligomers. Thus 987-1001 and 1025-1039 18S rRNA sequences are supposed to be involved in interaction with mRNA in the course of translation.
根据我们之前的研究结果，人类18S rRNA中的980 - 1061区域被选中，该结果表明烷基化mRNA类似物的交联位点位于此区域内。在本研究中，我们使用了10条与18S rRNA位置980 - 1061内各种重叠序列互补的15聚体DNA。通过用RNase H水解异源双链体中与相应探针结合的18S rRNA，证明了它们在所需rRNA序列上选择性结合的能力。只有4种探针能够与40S亚基结合，这表明相应的18S rRNA序列980 - 994、987 - 1001、1025 - 1039和1032 - 1046在亚基中是暴露的。没有一种探针抑制寡聚（U）信使与40S亚基的tRNA依赖性结合。然而，两种与18S rRNA序列987 - 1001和1025 - 1039互补的探针共价连接到40S亚基后，在无细胞系统中抑制了人80S核糖体对聚（U）的翻译。复合物pAUG.40S.Met - tRNA.eIF - 2.GTP中信使三核苷酸的结合受到相同寡聚物的强烈影响。因此，18S rRNA序列987 - 1001和1025 - 1039被认为在翻译过程中参与与mRNA的相互作用。
