TB-RBP, also called translin, is a 27 kD protein that performs a range of important biological functions in the temporal and spatial expression of mRNA. The protein binds a specific region, the Y and H elements, of selected mRNAs for temporary silencing during sperm cell development. Binding of mRNA is now known to be controlled by guanine nucleotide binding. TB-RBP also serves as an adapter to motor proteins to facilitate mRNA transport among developing male germ cells, and along neurons in the brain. TB-RBP can also bind ssDNA in the nucleus and has been associated with DNA breaks and oncogene translocation in leukemias. Our proposed research should produce important insights into the mechanism of mRNA recognition, silencing, and transport, as well as an understanding of how the protein facilitates oncogenic gene translocation. We have produced the first X-ray structure for TB-RBP, or any homologue, from a 2.7 A map. The protein, which exhibits a novel fold, assembles into an octamer with a substantial central cavity that is hypothesized to bind a compactly folded RNA element. We propose to complete our model and to refine it to the limits of diffraction. We also have soaked GDP into our crystals and co-crystallized the protein with GTP for analysis of the conformational changes governed by GTP. We propose to examine the mode of RNA and DNA binding by co-crystallization with appropriate nucleic acid sequence; this may well exhibit a novel type of nucleic acid binding. The protein TRAX has also been expressed. It is a homologue of TB-RBP and forms multimers with TB-RBP causing it to release silenced RNA. We propose to crystallize the native monomer and the TB-RBPTRAX heterodimer. In addition, g-actin and KIF3 are parts of motor systems known to bind TB-RBP. Co-crystallization and biochemical studies will be undertaken to define how these molecular machines assemble. In collaboration with a group at the University of Pennsylvania Medical School, we will make and characterize key site-directed mutants to explore all these functions. The goal is to elucidate the details of TB-RBP assembly, nucleic acid binding, GTP effector binding, and interactions with TRAX and motor proteins. Together, this work should elucidate the molecular structure and the mode of action and regulation of an important new class of DNA/RNA-binding proteins of great interest to the scientific and medical community.
Showing the most recent 10 out of 14 publications
