RNA-protein interactions are essential for many regulatory and cellular processes, including transcription, RNA splicing, and translation. Although these interactions play crucial roles in the cell, only a modest amount is known about the details of sequence-specific RNA recognition, in part because RNA can fold into complicated tertiary structures. It is clear that both RNA structure and base sequence play important roles in recognition. The study of RNA-protein interactions has been somewhat simplified by the discovery of a few common motifs in RNA-binding proteins. One of these, the arginine-rich motif, is found in the HIV Tat protein. Tat uses a single arginine residue, in the midst of a region of basic amino acids, to specifically recognize the TAR RNA hairpin. Arginine appears to recognize a particular structural feature of TAR, probably simultaneously hydrogen bonding to two phosphates on the backbone and to a base in the major groove. The interaction of arginine with two adjacent phosphates, termed an "arginine fork", provides a possible mechanism for a protein to indirectly read out a base sequence by recognizing a sequence- dependent RNA structure. Other modes of binding have been seen with arginine and the guanosine-binding site of a group 1 intron, and with arginines of tRNA synthetases involved in tRNA recognition. This proposal will use the specific binding of arginine as a tool to probe RNA structure and folding and to define arginine-RNA interactions that may be important in RNA-protein recognition. The finding that short arginine-containing peptides, or even free arginine, can bind specifically to RNAs makes it possible to ask which features of RNA structure cause it to adopt specific arginine-binding conformations. RNA selection methods will be used to identify RNAs from random mixtures that specifically bind arginine. Groups on the RNA bases and backbone that interact with arginine will be mapped by chemical modification and mutagenesis experiments. Possible conformational changes in the RNAs, which have been seen in other RNA-protein interactions, will be examined by circular dichroism and NMR experiments. the role of surrounding charged amino acids within the arginine-rich motif will be determined by mutagenesis in the Tat-TAR system and by structural comparisons of RNAs selected with peptides. Selection experiments will be used to ask whether DNAs can adopt specific arginine-binding conformations and whether these structures are similar to or different than the selected RNAs. RNAs will be prepared to determine the detailed three-dimensional structures of interesting RNAs and arginine-RNA complexes by NMR and X- ray crystallography. It is anticipated that the studies proposed here will shed light on fundamental features of RNA-protein recognition, RNA folding, and RNA structure, and thus be important for understanding mechanisms of cellular regulation.