Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNAPhe (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNAPhe (yeast) intramolecularly crosslinked in aqueous solution.

Abstract

Intramolecular crosslinking of yeast phenylalanine tRNA in aqueous solution with rigid, variable-length crosslinking reagents, which we call "molecular rulers," has given results in reasonable agreement with the crystal structure. Chlorambucilyl-[3H]phenylalanyl-tRNAPhe crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[3H]phenylalanyl-tRNAPhe crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 A long, including chlorambucil and phenylalanine; the sites that it reached are 60 A distant from the 3' OH (in the case of G-20) or 80 A distant (in the case of Y-37) in the crystal structure of tRNAPhe. The close agreement between the length of the reagent and the distance of G-20 from the 3' OH in the crystal structure illustrates the rigidity of the tRNAPhe molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-A-long reagent to crosslink to a site, Y-37, that is 80 A distant from the 3' OH in the crystal structure appears to illustrate the flexibility of both the 3' A-C-C-A terminus and the anticodon stem and loop, with respect to the tRNA molecule. These observations demonstrate the utility of oligoproline-based crosslinking reagents as rigid, variable-length molecular rulers for biological macromolecules in solution.