Molecular rulers for measuring RNA structure: sites of crosslinking in chlorambucilyl-phenylalanyl-tRNA^(Phe) (yeast) and chlorambucilyl-pentadecaprolyl-phenylalanyl-tRNA^(Phe) (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-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked intramolecularly at G-71 and A-73, whereas chlorambucilyl-pentadecaprolyl-[^(3)H]phenylalanyl-tRNA^(Phe) crosslinked at G-20 and Y-37. The pentadecaprolyl reagent was predicted to be 62 Å long, including chlorambucil and phenylalanine; the sites that it reached are 60 Å distant from the 3' OH (in the case of G-20) or 80 Å distant (in the case of Y-37) in the crystal structure of tRNA^(Phe). 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 tRNA^(Phe) molecule in the dihydrouridine loop region at the corner of the molecule. The apparent ability of the 62-Å-long reagent to crosslink to a site, Y-37, that is 80 Å 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.

Files

Additional details