A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.
A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.
复制标题
使用 5-溴尿苷取代的 RNA 进行特定的 UV 诱导 RNA 蛋白交联。
DOI：
10.1021/bi00239a030
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Uhlenbeck,OC
中科院分区：
文献类型：
--
作者：
Gott,JM;Willis,MC;Koch,TH;Uhlenbeck,OC
材料和方法RNA。RNA片段是通过T7RNA聚合酶从合成的DNA模板中体外转录得到的(Milligan等人，1987)。转录反应包括40 mM Tris-HCl(37℃，pH 8.1)，1 mM亚精胺，5 mM二硫苏糖醇，50 mg/mL牛血清白蛋白(BSA)，0.1%(v/v)Triton X-100，80 mg/mL聚乙二醇(MR 8000)。在40-mL的转录反应中制备标记的RNA，3个NTP(使用UTP或BrUTP)、0.25 mM[a-32P]NTP(5 MCI)、6 mM氯化镁、100-400 nM模板和0.05-0.1 mg/mLT7 RNA聚合酶各1 mM。核苷酸，包括5-BrUTP，购自Sigma。用20%变性聚丙烯酰胺凝胶电泳法纯化RNA片段，用90 mM三硼酸/2 mM EDTA洗脱，0.1M Tris-HCl(PH 8)/L mM EDTA洗脱过夜，在0.4M醋酸铵存在下乙醇沉淀。外壳蛋白表达系统。利用Studier和Moffat(1986)的过表达系统分离了大量的噬菌体外壳蛋白。质粒Pt7-2的衍生物(美国生化公司)含有832bp的Nrul-Nael片段，编码噬菌体基质蛋白的3‘端，以及在噬菌体T7启动子控制下的整个外壳蛋白基因。上游序列(Nrul-Xbal)来自pSIU510(Parker&Precup，1986)，外壳蛋白基因(Xbal-Nael)来自pCOAT184(Berkhout，1986)。用pTCT5转化BL21(DE3)，在lac启动子的控制下，BL21(DE3)含有整合到细菌基因组中的T7噬菌体RNA聚合酶基因(Studier&Moffat，1986)。异丙基/3-D-硫代半乳糖苷(IPTG)诱导产生高水平的
Materials and Methods RNAs. RNA fragments were prepared by in vitro transcription from synthetic DNA templates by T7 RNA polym-erase (Milligan et al., 1987). Transcription reactions contained 40 mM Tris-HCl (pH 8.1 at 37 C), 1 mM spermidine, 5 mM dithiothreitol, 50 Mg/mL bovine serum albumin (BSA), 0.1%(v/v) Triton X-100, and 80 mg/mL polyethylene glycol)(Mr 8000). Labeled RNAs were prepared in a 40-mL transcription reaction with 1 mM each of three NTPs (using either UTP or BrUTP), 0.25 mM [a-32P] NTP (5 MCi), 6 mM MgCl2, 100-400 nM template, and 0.05-0.1 mg/mL T7 RNA polymerase. Nucleotides, including 5-BrUTP, were purchased from Sigma. RNA fragments were gel-purified by electro-phoresis on 20% denaturing polyacrylamide gels run in 90 mM Tris-borate/2 mM EDTA (TBE), cut out, eluted overnight in 0.1 M Tris-HCl (pH 8)/l mM EDTA, and ethanol-precipitated in the presence of 0.4 M ammonium acetate. Coat Protein Expression System. The overexpression system of Studier and Moffat (1986) was used to isolatelarge amounts of phage coatprotein. A derivative of plasmid pT7-2 (US Biochemical Corp.) was used which contains a 832 bp Nrul-Nael fragment encoding the 3'end of the phage matu-ration protein and the entire coat protein gene under the control of a phage T7 promotor. The upstream sequences (Nrul-Xbal) were derived from pSIU510 (Parker & Precup, 1986), while the coat protein gene (Xbal-Nael) was subcloned from pCOAT184 (Berkhout, 1986). This plasmid, pTCT5, was used to transform BL21 (DE3) which contains a single copy of the bacteriophage T7 RNA polymerase gene integrated into the bacterial genome (Studier & Moffat, 1986) under control of the lac promotor. Induction with isopropyl/3-D-thiogalactopyranoside (IPTG) resulted in high levels of