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 approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding 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 calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.
Golden
B L
BL
Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder 80309-0215, USA.
Gooding
A R
AR
Podell
E R
ER
Cech
T R
TR
D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli; D005818:Genetic Engineering / Q000379:methods*; D007065:Idoxuridine / Q000031:analogs & derivatives / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010316:Particle Size; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics; D013768:Tetrahymena / Q000235:genetics
D016337:RNA, Catalytic; D016053:RNA, Protozoan; C035991:5-iodouridine; D007065:Idoxuridine