X-RAY CRYSTALLOGRAPHY OF LARGE RNAS - HEAVY-ATOM DERIVATIVES BY RNA ENGINEERING

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than similar to 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Gate JH et al., 1996, Scie nce 273:1678-1685). To incorporate iodouridine, a two-piece RNA was co nstructed. The 5' segment, containing the majority of the molecule, wa s transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A syntheti c 5-iodouridine-containing RNA corresponding to the remainder of the s equence was then annealed to the transcribed piece of RNA. The resulti ng RNA appeared structurally and functionally sound as judged by nonde naturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different p ositions crystallized under the same conditions as the native RNA, yie lding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calc ulated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structure d RNA molecules.