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

成熟的5S RNA有五个茎连接并由环终止；主要的 转录本有一条3‘的单股尾巴，可通过加工去除。 由环D、茎IV和环E组成的内部区域是可有可无的 以供处理。我们已经在果蝇5S RNA中进行了多次替换 映射序列和结构对加工的影响，以及 解释了散布在茎中的加工元素的分布 I、II、III以及循环B和C两种方式。首先，顺序发生变化， 当结合时刺激加工合作；我们建议加工 蛋白质与5S RNA(远程协作)进行多次接触。 其次，加工不良的序列变体聚集在茎的中心 I和II，两侧是削弱碱基配对的位置变化 提高加工效率。我们建议多肽臂伸入FORM环 A产生茎I，从环A产生茎II，以形成所需的中心 合同(呼吸模式)。 我们建议研究远程合作和螺旋呼吸 通过分离5S RNA中涉及的蛋白质来实现茎/环连接 正在处理。一旦分离出加工蛋白，我们将评估 它们与野生型和变异型5S RNA的相互作用部位 脚印。茎I、II、III以及环B和C中的蛋白质接触将 坚持远程合作；这种联系应该被削弱 或在处理减少的序列变体中缺失。 我们将检测野生型和变异型5S RNA结构以评估AAT 呼吸模型。改进加工的替代产品应该增加 在以下情况下探查进入中央干收缩的途径 杆/环连接。最后，随机化/处理大小选择 实验将使我们能够分析序列变体的多种组合 用于通过定点突变进行不切实际的处理。 这些研究将有助于我们理解核酸-- 使用稳定的小RNA作为加工底物的蛋白质相互作用。

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.