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.