Malygin A A, Graĭfer D M, Zenkova M A, Mamaev S V, Karpova G G
Mol Biol (Mosk). 1992 Mar-Apr;26(2):369-77.
Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit. The sites of the reagents attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 derivative and within 976-1164 for (pU)3 one. These sites are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.
带有通过磷酰胺键连接到5'-磷酸基团上的4-(N-2-氯乙基-N-甲基氨基)苄基甲胺残基的5'-[³²P]标记的(pU)₃和(pU)₆衍生物，被用于对人胎盘80S核糖体进行亲和标记。这些试剂具有正常的编码特性，并通过与P位点（对于(pU)₃衍生物而言）或A和P位点（对于(pU)₆衍生物而言）上的同源苯丙氨酰-tRNA(Rhe)的密码子-反密码子相互作用，固定在核糖体mRNA结合区域。发现这两种试剂仅修饰40S亚基。通过将修饰后的18S核糖体RNA与相应rDNA的限制性片段进行印迹杂交，确定了试剂与18S核糖体RNA的结合位点。发现对于(pU)₆衍生物，这些位点位于976 - 1057位置内；对于(pU)₃衍生物，位于976 - 1164位置内。这些位点大概位于真核小亚基rRNA二级结构的高度保守部分内。
