2'-mercaptonucleotide interference reveals regions of close packing within folded RNA molecules.
The 2'-hydroxyl group makes essential contributions to RNA structure and function. As an approach to assess the ability of a mercapto group to serve as a functional analogue for the 2'-hydroxyl group, we synthesized 2'-mercaptonucleotides for use in nucleotide analogue interference mapping. To correlate the observed interference effects with tertiary structure, we used the independently folding DeltaC209 P4-P6 domain from the Tetrahymena group I intron. We generated populations of DeltaC209 P4-P6 molecules containing 2'-mercaptonucleotides located randomly throughout the domain and separated the folded molecules from the unfolded molecules by nondenaturing gel electrophoresis. Iodine-induced cleavage of the RNA molecules revealed the sites at which 2'-mercaptonucleotides interfere with folding. These interferences cluster in the most densely packed regions of the tertiary structure, occurring only at sites that lack the space and flexibility to accommodate a sulfur atom. Interference mapping with 2'-mercaptonucleotides therefore provides a method by which to identify structurally rigid and densely packed regions within folded RNA molecules.
Schwans
Jason P
JP
Howard Hughes Medical Institute, Department of Biochemistry, The University of Chicago, Chicago, IL 60637, USA.
Cortez
Cecilia N
CN
Olvera
Joe M
JM
Piccirilli
Joseph A
JA
D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013816:Thermodynamics
D009711:Nucleotides; D013438:Sulfhydryl Compounds; D012313:RNA