POWRE: Dissecting an RNA-Protein Interaction VISITING RESEARCHER

9726943 White这是power访问研究员奖。首席研究员将在斯克里普斯研究所James R. Williamson博士的实验室进行为期一年的研究。选择主办实验室是因为。Williamson和White对RNA结构和蛋白质对RNA的识别有共同的兴趣，因为他们都在研究酵母L32系统。简而言之，酵母核糖体蛋白L32结合其未剪接或剪接的转录物来抑制剪接或翻译，因此是一种自调节蛋白。在过去的六年里，怀特博士的实验室发现，L32模型rna小至24个核苷酸以纳摩尔的亲和力与L32结合，使该系统成为结构研究的一个有吸引力的目标。Williamson实验室目前从事L32 RNA及其结合蛋白的核磁共振研究。提出的研究将提供更多的生化数据，以补充结构数据。具体解决的两个问题是潜在的非沃森-克里克对的氢键状态和RNA结合关键的氨基酸残基的定位。第一组实验将解决L32 RNA内部环中的氢键问题。L32 RNA采用茎-内环-茎结构，内环是蛋白质结合的关键，由嘌呤组成。先前的结构映射实验表明，该区域的一些碱基是堆叠的，但是特定的嘌呤N7位置可以进行化学修饰。一对并置的GA二核苷酸作为与内环相邻的潜在G:U对是保守的。RNA将在通常参与沃森-克里克氢键的碱基位置进行化学修饰。比较变性和折叠的L32 RNA将表明折叠后的RNA是否由于氢键作用而不受化学修饰的保护。这种引物延伸化学修饰的检测将是怀特实验室一项重要的、通用的新技术。其次，一种强大的新基因筛选将用于确定哪些L32氨基酸与特定的RNA核苷酸接触。双质粒系统将用于寻找抑制蛋白，恢复与突变rna的结合。报告质粒将编码-半乳糖苷酶基因上游L32结合位点的序列。缺乏蛋白质结合的RNA序列将被选择。随机突变的L32蛋白的基因将在另一个质粒上表达，即抑制因子。如果该蛋白变体不能与L32/ β -半乳糖苷酶转录物结合，则会产生β -半乳糖苷酶。然而，如果蛋白质确实与嵌合转录物结合，这种结合将阻断通往核糖体结合位点的通道，并且几乎不会产生任何蛋白质。抑制因子将通过蓝/白菌落颜色测试和测量抑制比来识别。最终，将特定的RNA核苷酸与特定的氨基酸连接起来的图谱将产生。与此同时，Williamson小组将致力于通过核磁共振来解决L32蛋白及其RNA复合物的结构。这种基因筛选和结构确定的双管齐下的方法应该是强有力的。当怀特医生回到布林茅尔时，屏幕的设置将为她的小组带来新的技术。怀特博士将有机会在斯克里普斯研究所进行为期一年的访问研究，这将使他能够在L32c系统的生物化学方面取得进展，学习新技术，与结构生物学家合作，并与斯克里普斯非常令人兴奋的科学界进行互动。* * *

9726943 White This is a POWRE Visiting Researcher award. The Principal Investigator will spend a full sabbatical year of research at the Scripps Research Institute in the laboratory of Dr. James R. Williamson. The host laboratory was chosen because Drs. Williamson and White have shared interests in RNA structure and RNA recognition by proteins, and because they are both working on the yeast L32 system. Briefly, yeast ribosomal protein L32 binds to its unspliced or spliced transcript to inhibit splicing or translation and thus is an autoregulatory protein. During the past six years, Dr. White's laboratory has discovered that model L32 RNAs as small as 24 nucleotides bind L32 at nanomolar affinities, making this system an attractive target for structural studies. The Williamson laboratory is currently engaged in NMR studies of L32 RNA and its binding protein. The proposed research will provide more biochemical data that will complement the structural data. The two questions specifically addressed are hydrogen bonding status of potential non Watson-Crick pairs and localization of amino acid residues critical for RNA binding. The first set of experiments will address the question of hydrogen bonding in the L32 RNA's internal loop. The L32 RNA adopts a stem-internal loop-stem structure and the internal loop is critical for protein binding and is composed of purines. The previous structure-mapping experiments show that some bases in this region are stacked, but that particular purine N7 positions are accessible to chemical modification. A pair of juxtaposed GA dinucleotides is conserved as is a potential G:U pair bordering the internal loop. The RNA will be chemically modified at the base positions that normally participate in Watson-Crick hydrogen bonding. Comparisons of denatured and folded L32 RNA will indicate whether the folded RNA is protected from chemical modification by virtue of hydrogen bonding. This detection of chemical modification by primer extension will be an import ant and versatile new technique for the White laboratory. Secondly, a powerful new genetic screen will be used to determine which L32 amino acids contact particular RNA nucleotides. A two-plasmid system will be used to find suppressor proteins that restore binding to mutant RNAs. The reporter plasmid will encode the sequence for the L32 binding site just upstream of a beta-galactosidase gene. RNA sequences deficient in protein binding will be chosen. The gene for the randomly mutagenized L32 protein will be expressed on the other plasmid, the repressor. If the protein variant fails to bind to the L32/beta-galactosidase transcript, then beta-galactosidase will be produced. However, if the protein does bind the chimeric transcript, the binding will block access to the ribosome binding site and little if any of the protein will be produced. Suppressors will be identified by a blue/white colony color test and by measuring repression ratios. Ultimately a map which links a particular RNA nucleotide with a particular amino acid will result. At the same time, the Williamson group will be working to solve the structure of the L32 protein and its RNA complex by NMR. This two-pronged approach of a genetic screen and structure determination should prove to be a powerful one. Setting up of the screen will bring new techniques to Dr. White's group when she returns to Bryn Mawr. The opportunity to undertake a full year as a Visiting Researcher at the Scripps Research Institute which is provided by this POWRE award will enable Dr. White to make progress on the biochemistry of the L32c system, learn new techniques, collaborate with structural biologists, and interact with the very stimulating scientific community at Scripps. ***