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

5S RNA，蛋白质合成所需的核糖体大亚基的小RNA 合成，具有保守的五个结构域茎环二级结构。 像 大部分真核RNA，D.黑腹果蝇5S RNA在 转录。 135 nt的初级转录物从其3 '端去除15 nt， 结束加工。 这一提议涉及5S RNA的结构基础 处理. 5S RNA加工相对不受核苷酸变化的影响 在加工位点下游的+120处，并且通过从 另一方面，在茎III中的缺失，移除茎IV-V的所有部分， IV-V，并破坏+1-118碱基对，即第一对核苷酸 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 处理. 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5S RNA将被转录 与T7 RNA聚合酶，并使用果蝇进行测试， 加工提取物的能力进行处理。 三组突变将被引入到顶部或底部的 螺旋茎破坏碱基配对，或进入顶部和底部， 恢复它。成对的删除和插入将被用来改变 螺旋的长度 本文还分析了长度和顺序的影响 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 和碱基配对结构对果蝇信息加工能力的影响 黑腹果蝇5S 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.