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: 6107222
• 负责人: LEVINGER LOUIS
• 金额: $ 20.71万
• 依托单位: YORK COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107222
• 关键词: DNA footprinting; Drosophilidae; RNA biosynthesis; affinity chromatography; chemical synthesis; endonuclease; gel mobility shift assay; gene mutation; genetic regulation; genetic transcription; ion exchange chromatography; nucleic acid reconstitution; nucleic acid sequence; nucleic acid structure; oligonucleotides; polymerase chain reaction; posttranscriptional RNA processing; site directed mutagenesis; small nuclear RNA

Mature 5S RNA has five stems connected and terminated by loops; the primary transcript has a 3' single stranded tail which is removed by processing. The internal domain consisting of loop D, stem IV and loop E is dispensable for processing. We have made numerous substitutions in Drosophila 5S RNA to map the influence of sequence and structure on processing, and interpreted the distribution of processing elements dispersed through stems I, II, III and loops B and C in two ways. First, sequence changes which stimulate processing cooperate when combined; we suggest that a processing protein makes multiple contacts with 5S RNA (long range cooperation). Second, poorly processed sequence variants cluster in the center of stems I and II, flanked by positions where changes which weaken base pairing improve processing. We suggest that a polypeptide arm reaches in from loop A yields stem I and from loop A yields stem II to make required central contracts (the breathing model). We propose to investigate long range cooperation and helix breathing at stem/loop junctions by separating the proteins involved in 5S RNA processing. Once the processing proteins are isolated, we will assess their sites of interaction with wild type and variant 5S RNA by footprinting. Protein contacts in stems I, II, III and loops B and C would be consistent with long range cooperation; such contacts should be weakened or absent from sequence variants with reduced processing. We will probe wild type and variant 5S RNA structure to evaluate athe breathing model. Substitutions which improve processing should increase probe access to central stem contracts in the event of breathing at stem/loop junctions. Finally, a randomization/processing size selection experiment will enable us to analyze many combinations of sequence variants for processing which would be impractical by site-specific mutagenesis. These investigations will contribute to our understanding of nucleic acid- protein interactions by using a small stable RNA as a processing substrate.

成熟的5S RNA具有五个茎连接并通过环终止。主要 转录本具有3'单链尾巴，通过处理去除。 由循环D，茎IV和环E组成的内部域是可分配的 用于处理。 我们在果蝇5S RNA中做出了许多替代品 绘制序列和结构对处理的影响，以及 解释了通过茎分散的处理元件的分布 I，II，III和LOOP B和C有两种方式。 首先，序列改变了 合并时刺激处理合作；我们建议处理 蛋白质与5S RNA（远程合作）进行了多次接触。 其次，处理不良的序列变体聚集在茎的中心 i和ii，两侧是位置，其中变化会削弱基础配对 改善处理。 我们建议从循环到达多肽臂 收益茎i和从环的a产量茎ii以使所需的中央 合同（呼吸模型）。 我们建议调查远程合作和螺旋呼吸 通过分离涉及5s RNA的蛋白质的茎/环连接 加工。 一旦孤立了加工蛋白，我们将评估 他们与野生型和变体5S RNA相互作用的位点 足迹。 茎I，II，III和LOOP B和C中的蛋白质接触将 与远程合作保持一致；这样的联系应削弱 或从序列变体中不存在，并且处理减少。 我们将探测野生型和变体5S RNA结构以评估ATHE 呼吸模型。 改善处理的替换应增加 如果呼吸呼吸，请访问中央STEM合同 茎/循环连接。 最后，随机化/处理尺寸选择 实验将使我们能够分析序列变体的许多组合 用于处理，这是由位点特异性诱变而不切实际的。 这些研究将有助于我们对核酸的理解 通过使用小稳定RNA作为加工底物，蛋白质相互作用。