PROTEINS INVOLVED IN D MELANOGASTER 5S RNA PROCESSING

描述(改编自申请者摘要)：果蝇 黑素胃泌素5S核糖体RNA作为前体转录，由 将15个核苷酸从其3‘端移除并整合到大的核糖体中 亚单位。果蝇S100提取物对5S RNA的体外加工 乙二胺四乙酸乙二酯、蛋白酶K消化或 加热到37摄氏度，表明对二价阳离子和 蛋白质。将提取物与核糖核酸酶预孵化，然后进行适当的 核糖核酸酶抑制剂不影响加工，表明内源 黑腹果蝇5S可能不直接需要S100 RNA RNA处理。获得了135个核苷酸底物5S RNA的初级转录本 来自使用果蝇黑腹果蝇5S RNA的T7 RNA聚合反应 与T7启动子连锁的基因。这种结构使调查员能够 删除成熟5S RNA的结构域并测试这些突变RNA的 被处理的能力。成熟的5S RNA由5个保守的 茎环结构域。茎IV的全部和茎V的一半对于 处理，而处理身份元素广泛分散 在5S RNA的其余部分。这项提议涉及分馏的问题 对S100提取物进行了研究，以考察加工和5S RNA结合成分。 提取液中的蛋白质可以通过离子交换和亲和作用分离出来。 层析法。加工反应将通过孵化进行 柱馏分与5S RNA初级转录本的混合物 变性聚丙烯酰胺凝胶电泳法。忠实的3‘端阵型 已经通过制备工艺进行了研究，二维RNaseT1 指纹图谱和次级核酸酶分析。绑定实验 柱分数将使调查者能够将 5S RNA-蛋白质相互作用与加工活性。这些实验 包括非变性聚丙烯酰胺凝胶移位、单一 末端标记的5S RNA和放射性光亲和交联剂 标记的RNA以可视化结合蛋白。拟议的研究 剖析了一种功能重要且结构复杂的RNA-蛋白质 互动。

DESCRIPTION (Adapted from the applicant's abstract): Drosophila melanogaster 5S ribosomal RNA is transcribed as a precursor, processed by removal of 15 nt form its 3' end and incorporated into the large ribosomal subunit. In vitro 5S RNA processing using an S100 extract form Drosophila melanogaster culture cells is prevented by EDTA, proteinase K digestion or heating to 37 degrees C, suggesting dependence on divalent cations and proteins. Preincubation of extract with RNases followed by the proper RNase inhibitors does not affect processing, indicating that endogenous S100 RNA is probably not directly required for Drosophila melanogaster 5S RNA processing. 135 nt substrate 5S RNA primary transcript was obtained from a T7 RNA polymerase reaction using a Drosophila melanogaster 5S RNA gene linked to a T7 promoter. This construction allowed the investigator to delete domains of mature 5S RNA and test these mutant RNAs for their ability to be processed. Mature 5S RNA consists of five conserved stem-loop domains. All of stem IV and half of stem V are dispensable for processing, while processing identity elements are widely dispersed throughout the rest of 5S RNA. This proposal concerns the fractionation of the S100 extract to investigate processing and 5S RNA binding components. Proteins from the extract can be separated by ion exchange and affinity chromatography. Processing reactions will be performed by incubating mixtures of column fractions with 5S RNA primary transcript and analyzed by denaturing polyacrylamide gel electrophoresis. Faithful 3' end formation has been investigated by preparative processing, two dimensional RNase T1 fingerprinting and secondary nuclease analysis. Binding experiments with column fractions will enable the investigator to correlate specificity of 5S RNA-protein interactions with processing activity. These experiments include nondenaturing polyacrylamide gel shifts, footprinting with single endlabeled 5S RNA, and photoaffinity crosslinking with radioactively labeled RNA to visualize the binding proteins. The proposed research dissects a functionally important and RNA structurally complex RNA-protein interaction.