A photo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme.
We have identified an essential UV-sensitive tertiary structure domain within the hairpin ribozyme. Irradiation at 254 nm produces two cross-linked RNA species that are resolved from the unmodified structure by denaturing gel electrophoresis. One cross-link forms at high efficiency and maps between nucleotides G21 and/or A22 and U41, all essential bases located within an internal loop joining helices 3 and 4. A second cross-link forms between nucleotides A20 and U42 as a result of ribozyme dimerization at concentrations greater than 0.5 microM. Both cross-linked species retain cleavage activity and so presumably reflect catalytically proficient structures of the ribozyme. Formation of the intramolecular cross-link is independent of Mg2+ and substrate and is blocked by base substitutions within the reactive domain that inhibit catalysis. A 36-nt RNA fragment containing the photoreactive domain but lacking the substrate binding domain also cross-links with high efficiency and maps between G21 and U41, as observed with the intact molecule. The sequence and cross-linking sites of the UV-sensitive internal loop are strikingly similar to those found in several other RNA molecules, including loop E of 5S rRNA. These results suggest that the loop E-like structure may be a common RNA folding domain that is utilized in a variety of functionally important RNA molecules.
Butcher
S E
SE
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Burke
J M
JM
D001483:Base Sequence; D002384:Catalysis; D008274:Magnesium; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D016337:RNA, Catalytic; D008274:Magnesium