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一小部分RNA 合成具有保守的五个域茎环二级结构。 喜欢 大多数真核RNA，D。Melanogaster5s RNA在 转录。 135 nt初级笔录已从其3'中删除15 nt 结束处理。 该建议涉及5S RNA的结构基础 加工。 5s RNA处理相对不受核苷酸的变化影响 从+120的处理地点下游，并通过删除30 nt STEM IV-V。 另一方面，删除词干III，删除所有词干 IV-V和 +1-118碱基对的破坏，第一个成对的核苷酸 在STEM I中，都干扰了处理。 在此，我建议彻底研究5S RNA所需的域 加工。 与T7 RNA相关的5S RNA基因的位置定向诱变 将执行聚合酶启动子。 突变5S RNA将被转录 使用T7 RNA聚合酶，并使用果蝇Melanogaster进行了测试 处理提取物的处理能力。 三个突变集将引入到顶部或底部 螺旋茎破坏基础配对，或者在顶部和底部到达 恢复它。 将进行配对的删除和插入以更改 螺旋长度。 长度和序列的效果也将进行分析 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 果蝇的加工能力和基础配对结构 Melanogaster 5s RNA。