Directly photocrosslinked nucleotides joining transfer RNA to aminoacyl-tRNA synthetase in methionine and tyrosine systems

Abstract

We have used ultraviolet photocrosslinking and 32P post-labeling to help define the contact surface between transfer RNAs and aminoacyl-tRNA synthetases for the methionine and tyrosine systems. Photocrosslinking between tRNAs and synthetases is shown to occur only in cognate complexes. The increased sensitivity of our procedures reduces the amounts of interacting macromolecules and permits lower ultraviolet light doses, thereby minimizing radiation damage. These procedures have detected crosslinks only within the 3′-terminal RNase T 1 fragments in yeast tRNA i Met and Escherichia coli tRNA 2 Tyr; and although the photoadducts were unstable, we have identified the crosslinked nucleotides. These crosslinks occur at positions C74 and A76 in yeast tRNA i Met and position U64 in E. coli tRNA 1&2 Tyr (conventional tRNA numbering system of Gauss & Sprinzl, 1981). This work demonstrates that even labile photocrosslinks can be exploited for mapping crosslinked nucleotides.