DEFINING THE ORIENTATION OF THE HUMAN U1A RBD1 ON ITS UTR BY TETHERED-EDTA(FE) CLEAVAGE

The N-terminal RNA binding domain of the human U1A protein (RBD1) spec ifically binds an RNA hairpin of U1 snRNA as well as two internal loop s in the 3' UTR of its own mRNA. Here, a single cysteine has been intr oduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA -5-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivat ive is used to generate hydroxyl radicals to cleave the proximal RNA s ugar-phosphate backbone in the RNA-RED complexes, RBD1(K20C)-EPD-Fe cl eaves the 5' strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, aga in centered on the 5' side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orie nt the two proteins bound to the UTR, and provide direct biochemical e vidence for the proposed model of the RBD1:UTR complex.