Analysis of Protein-Nucleic Acid Interactions Using Phosphor Imaging Technology

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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.