Detection of high-affinity intercalator sites in a ribosomal RNA fragment by the affinity cleavage intercalator methidiumpropyl-EDTA-iron(II).
Detection of high-affinity intercalator sites in a ribosomal RNA fragment by the affinity cleavage intercalator methidiumpropyl-EDTA-iron(II).
复制标题
通过亲和裂解嵌入剂甲锭丙基-EDTA-铁 (II) 检测核糖体 RNA 片段中的高亲和力嵌入剂位点。
DOI：
10.1021/bi00340a016
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Draper,DE
中科院分区：
文献类型：
--
作者：
Kean,JM;White,SA;Draper,DE
材料和方法化学品。所有溶液均用通过Millipore“Milli-Q”装置纯化的水配制。MPE是Peter Dervan给我的礼物。酵母tRNAPhe购自Sigma，溴化乙锭购自CalBiochem，硫酸铵铁（II）购自Aldrich，多核苷酸购自Pharmacia和PL Biochemicals，RNA连接酶、T、核糖核酸酶和聚丙烯酰胺凝胶试剂购自BRL，32 P核苷酸购自阿默舍姆。标记的RNA。在D’Alessio（1982）描述的条件下，用RNA连接酶和胞苷3，5，-[32 P]二磷酸标记tRNAPhe的3’末端。将反应混合物在40或85 cm测序凝胶上运行，并通过放射自显影定位全长tRNA条带并切下。如前所述（Kean &德雷珀，1985），从凝胶中提取RNA并提取苯酚。将tRNA储存在0.2MNaCl和50 mMTris（pH7.6）中，温度为-70 ℃，使用前在65 ℃复性5分钟. F61 RNA是来自大肠杆菌16 S核糖体RNA的345个碱基的片段，覆盖序列中的碱基525-869（Noller & Woese，1981）。它是通过杂交选择方法制备的;其制备和5 '和3'末端标记在前面的论文中有详细描述（Kean &德雷珀，1985）。
Materials and MethodsChemicals. All solutions were made up with water purified through a Millipore “Milli-Q” apparatus. MPE was a gen-erous gift from Peter Dervan. Yeast tRNAPhe was purchased from Sigma, ethidium bromide was from CalBiochem, am-monium iron (II) sulfate was from Aldrich, polynucleotides were fromPharmacia and PL Biochemicals, RNA ligase, T, ribonuclease, and polyacrylamide gel reagents were from BRL, and 32P nucleotides were from Amersham. Labeled RNAs. tRNAPhe was 3'end labeled with RNA ligase and cytidine 3,, 5,-[32P] bisphosphate under the conditions described by D’Alessio (1982). The reaction mix was run on a 40-or 85-cm sequencing gel and the full-length tRNA band located by autoradiography and cut out. RNA was extracted from the gel and phenol extracted as described previously (Kean & Draper, 1985). The tRNA was stored in 0.2 M NaCl and 50 mM Tris, pH 7.6, at-70 C, and renaturedfor5 min at 65 C before use. F61 RNA is a 345-base fragment from the Escherichia coli 16S ribosomal RNA, covering bases 525-869 in the sequence (Noller & Woese, 1981). It was made by a hybridization Selection method; its preparation and 5'and 3'end labeling are described in detail in the preceding paper (Kean & Draper, 1985).