NSF Award
9206873
A zinc-finger is an independent polypeptide structural unit that is shaped by the co-ordination of a zinc atom. Hundreds of genes for potential zinc finger proteins have been identified in the genomes of eukaryotes. Yet the function of only a tiny fraction of these is known or can be predicted. To learn more about the recognition of RNA by zinc finger proteins Xenopus TFIIIA and p43 will be altered by recombinant means. These proteins have a similar organization of nine zinc fingers, yet TFIIIA binds specifically to RNA and DNA, whereas p43 binds only to RNA. Mutant zinc finger proteins will be constructed in which amino acids or groups of amino aids from DNA-binding zinc fingers will be replaced with amino acids from the corresponding position in RNA-bindings zinc fingers and their affinity for RNA and DNA will be compared. As a preliminary to designing sequence-specific RNA-binding proteins, mutant proteins will be synthesized in which the order of zinc fingers has been changed. To identify the RNA substrates of these mutants, oligonucleotides containing a random sequence will be transcribed to produced a population of RNA substrates. RNA molecules that bind to the mutant proteins will be selected, amplified and sequenced. %%% Many essential cellular functions are controlled through proteins that bind to RNA or DNA. One class of these proteins contains a protein structure call a 'zinc finger' -- a loop of amino acids shaped into a finger-like structure by the coordination of a zinc atom. Zinc fingers mediate protein binding to DNA and RNA. However, the molecular features that allow some zinc fingers to distinguish RNA and others DNA are unknown. This research seeks to understand the molecular basis for selectivity of zinc finger containing proteins for RNA or DNA. Two zinc finger proteins, TFIIIA and p4, provide and excellent model system for this study, because these proteins have a similar organization of nine zinc fingers, yet TFIIIA binds to both RNA and DNA, whereas p43 binds only to RNA. Therefore, the differences and similarities between these two proteins should enable us to learn which features of the zinc finger are needed for interaction with RNA and which features of the zinc finger are needed for interaction with RNA and which DNA. Using recombinant DNA technology to synthesize hybrids of TFIIIA and p43 in which different zinc fingers or parts of zinc fingers have been combined, the specificity and affinity of individual zinc fingers for RNA and DNA will be measured. These studies will lay the groundwork for designing protein molecules that bind to specific RNA sequences or structures. Such molecules could form the basis for a new class of therapeutic agents that may be able to block RNA function, for example by inhibiting translation of a specific messenger RNA into protein.
