Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex.
We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifying reagents dimethylsulfate, 1-cyclohexyl-N'-[2-(N-methylmorpholino) ethyl-carbodiimide-p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2,12-dimethyl-3,7,11,17- tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon addition of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loops A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without inducing major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.
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; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000494:pharmacology; D016337:RNA, Catalytic / Q000737:chemistry* / Q000145:classification / Q000187:drug effects; D013329:Structure-Activity Relationship; D013463:Sulfuric Acid Esters / Q000494:pharmacology
D009942:Organometallic Compounds; D016337:RNA, Catalytic; D013463:Sulfuric Acid Esters; C060331:1-cyclohexyl-3-(2-(4-morpholinyl)ethyl)carbodiimide; D003028:CME-Carbodiimide; C090497:nickel(II) (2,12-dimethyl-3,7,11,17-tetraazabicyclo(11.3.1)heptadeca-1(17),2,11,13,15-pentaene) perchlorate; D008274:Magnesium; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate