Analysis of Protein-Nucleic Acid Interactions Using Phosphor Imaging Technology

9318111战神核酸-蛋白质相互作用对遗传信息的正确表达和检索至关重要。 基因的选择性转录需要蛋白质和DNA之间的选择性相互作用。构建包含真核基因的蛋白质编码信息的信使RNA需要识别和去除内含子和RNA剪接，这是一个依赖于一组精心设计的RNA-RNA和RNA-蛋白质相互作用的过程。信使RNA翻译成蛋白质是由RNA和蛋白质组成的机器完成的，需要另一套精心设计的RNA-RNA和RNA-protein相互作用。mRNA水平的控制是调节基因表达的一种手段，通常由mRNA中的RNA结构元件控制。详细了解这些DNA-蛋白质，RNA-蛋白质和RNA-RNA相互作用如何以及何时建立，维持和分解对于理解遗传信息的表达至关重要。 一套用于测量核苷酸沿着核酸链的环境的强有力的“足迹”或“结构探测”技术得到了很好的发展，并且在核酸结构和功能的学生中普遍使用。这些方法依赖于检测所研究的核酸对能够与核酸反应的某种探针（化学物质或核酸酶）的可接近性的差异。使用各种技术在链上映射反应的位置，所有这些技术都需要直接或间接地对链进行放射性标记。放射性产物在凝胶上按大小分离，反应位点由放射性产物的大小计算。在特定位置的反应量可以通过特定尺寸的产物中存在的放射性物质的量来确定。例如，与核酸链上的特定位点结合的蛋白质将保护该链免受探针的攻击。在结合和未结合核酸的分离和混合后，代表受保护位点的凝胶带将含有较少的标记。这种一般方法的技术限制有两个方面。首先，通常难以分离足够量的功能相关的蛋白质-核酸复合物以获得可检测的信号，其次，一些相互作用是足够微妙的，以至于对探针的保护虽然有意义，但可能只是部分的，并且需要仔细定量信号。 这两种限制都源于一个原因：放射性凝胶暴露于照相胶片以检测和定量放射性信号的要求。 这些限制可以通过使用一种新技术来规避，即存储磷光体成像，而不是放射自显影。 荧光成像包括将足迹凝胶暴露在一种特殊的存储荧光屏上，荧光屏上结合了一种对辐射敏感的化合物。这种化合物通过吸收放射性粒子而转化。然后将屏幕插入激光扫描装置中，该激光扫描装置确定屏幕上每个坐标处存在的转化化合物的量。 这些数据被用来建立一个计算机文件，该文件使用灰度或颜色将每个位置的放射性物质的量表示为图像。研究者可以在监视器上检查图像，并使用图像分析软件对数据进行定量分析。这种技术比放射自显影术更敏感几个数量级，可以更好地检测信号，并且与照相胶片不同，磷光体对辐射的响应是线性的，可以精确定量。 该提案旨在通过购买和使用荧光成像技术分析可用的足迹数据，进一步加深我们对核酸-蛋白质复合物功能和RNA-RNA相互作用的理解。Noller和Puglisi小组将使用该装置研究翻译过程中的核糖体结构和功能。战神小组将利用它来研究小核RNA在剪接体组装和前信使RNA剪接中的作用。 Peck小组将需要这种技术来理解RNA聚合酶111转录复合物的组装和功能，西尔弗索恩小组将使用它来研究mRNA结构在mRNA稳定性调节中的作用。

9318111 Ares Nucleic acid-protein interactions are critical to the correct expression and retrieval of genetic information. Selective transcription of genes requires selective interactions between protein and DNA. Construction of a messenger RNA containing the protein coding information of eukaryotic genes requires the recognition and removal of introns and RNA splicing, a process dependent on an elaborate set of RNA-RNA and RNA-protein interactions. The translation of messenger RNA into protein is carried out by a machinery made of RNA and protein as well, requiring another elaborate set of RNA-RNA and RNA-protein interactions. Control of the level of mRNA is a means by which the expression of genes is regulated, often governed by RNA structural elements in the mRNA. Detailed knowledge of how and when these DNA-protein, RNA-protein, and RNA-RNA interactions are established, maintained and disolved will be essential for understanding the expression of genetic information. A set of powerful "footprinting" or "structure probing" techniques for measuring the environments of nucleotides along a nucleic acid chain is well developed and is in general use among students of nucleic acid structure and function. These approaches rely on detecting differences in accessibility of the nucleic acid under study to some probe (a chemical or nuclease), capable of reacting with nucleic acid. The positions of reaction are mapped on the chain using a variety of techniques, all of which require radioactive labeling of the chain either directly or indirectly. The radioactive products are separated by size on a gel and the sites of reaction are calculated from the size of radioactive products. The amount of reaction at a particular site can be determined by the amout of radioactive material present in products of a particular size. For example, a protein that binds to a specific site on a nucleic acid chain will protect the chain from attack by the probe. After separation and com parison of bound andunbound nucleic acid, gel bands representing protected sites will contain less label. The technical limitations of this general approach are two fold. First, it is often difficult to isolate sufficient amounts of functionally relevant protein-nucleic acid complexes to obtain detectable signals, and second, some interactions are sufficiently subtle that protection from probes, though meaningful, may be only partial, and require careful quantitation of signal. Both of these limitations arise from a single cause: the requirement for exposure of radioactive gels to photographic film for detection and quantitation of radioactive signal. These limitations can be skirted by the use of a new technology, storage phosphor imaging, rather than autoradiography. Phosphor imaging involves exposure of the footprinting gel to a special storage phosphor screen, on which a compound sensitive to radiation is bound. The compound is converted by absorption of a radioactive particle. The screen is then inserted into a laser scanning device that determines the amount of converted compound present at every coordinate on the screen. This data is used to build a computer file that expresses the amount of radiactive material at each position as an image using a grey scale or color. The investigator can inspect the image on a monitor and with the image analysis software, analyze the data quantitatively. This technique is orders of magnitude more sensitive than autoradiography allowing better detection of signal, and unlike photographic film the response of the phosphor to radiation is linear, allowing accurate quantitation. This proposal aims for a material extension of our understanding of the function of nucleic acid-protein complexes and RNA-RNA interactions through the analysis of available footprinting data by the purchase and use of phosphor imaging technology. The Noller and Puglisi groups will use the device in their studies of ribosome structure and func tion in the process of translation. The Ares group will use it in their studies of the role of small nuclear RNAs in spliceosome assembly and pre-messenger RNA splicing. The Peck group will require this technology for understanding the assembly and function of RNA polymerase 111 transcription complexes, and the Silverthorne group will use it to study the role of mRNA structure in the regulation of mRNA stability.