SEQUENCE AND STRUCTURE SIGNALS FOR 5S RNA PROCESSING IN DROSOPHILA MELANOGASTER

果蝇 5S RNA 处理的序列和结构信号

• 批准号: 3841625
• 负责人: LEVINGER LOUIS
• 金额: --
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3841625
• 关键词: Drosophilidae; RNA biosynthesis; chemical synthesis; endonuclease; gene mutation; genetic regulation; genetic transcription; nucleic acid sequence; oligonucleotides; polymerase chain reaction; posttranscriptional RNA processing; site directed mutagenesis; small nuclear RNA

5S RNA, a small RNA of the large ribosomal subunit required for protein synthesis, has a conserved five domain stem-loop secondary structure. Like most eukaryotic RNAs, D. melanogaster 5S RNA is not utilized directly after transcription. The 135 nt primary transcript has 15 nt removed from its 3' end by processing. This proposal concerns the structural basis for 5S RNA processing. 5S RNA processing is relatively unaffected by changes in the nucleotides downstream from the processing site at +120, and by deletion of 30 nt from stem IV-V. On the other hand, deletions in stem III, removing all of stem IV-V, and disruption of the +1-118 base pair, the first paired nucleotides in stem I, all interfere with processing. Herein I propose to thoroughly investigate the domains required for 5S RNA processing. Site-directed mutagenesis of 5S RNA genes linked to a T7 RNA polymerase promoter will be performed. Mutant 5S RNAs will be transcribed with T7 RNA polymerase and tested using a Drosophila melanogaster processing extract for their ability to be processed. Sets of three mutations will be introduced into the top or bottom of helical stems to disrupt base pairing, or into both top and bottom to restore it. Paired deletions and insertions will be made to alter the length of helixes. The effect of length and sequence will also be analyzed in each of the single stranded loops. By means of these experiments, we shall evaluate in detail the relative contribution of nucleotide sequence and base paired structure to the processing ability of Drosophila melanogaster 5S RNA.

5S RNA，蛋白质所需的大核糖体亚基的小RNA 合成，具有保守的五个结构域茎环二级结构。 喜欢 大多数真核生物 RNA，黑腹果蝇 5S RNA 不直接被利用 转录。 135 nt 初级转录本的 3' 端删除了 15 nt 通过处理结束。 该提案涉及 5S RNA 的结构基础 加工。 5S RNA 加工相对不受核苷酸变化的影响 加工位点下游+120处，并从其中删除30 nt 茎IV-V。 另一方面，删除茎III，除去所有茎 IV-V，以及+1-118碱基对（第一对核苷酸）的破坏 在干 I 中，所有干扰处理。 在此我建议彻底研究 5S RNA 所需的结构域 加工。 与 T7 RNA 连接的 5S RNA 基因的定点诱变 将进行聚合酶启动子。 突变的 5S RNA 将被转录 使用 T7 RNA 聚合酶并使用果蝇进行测试 加工提取物以提高其加工能力。 三个突变组将被引入到顶部或底部 螺旋茎破坏碱基配对，或进入顶部和底部以破坏碱基配对 恢复它。 将进行配对删除和插入以改变 螺旋的长度。 还将分析长度和序列的影响 在每个单链环中。 通过这些实验，我们 应详细评估核苷酸序列的相对贡献 碱基配对结构对果蝇加工能力的影响 黑腹果蝇 5S RNA。