The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent.
We describe the use of site specific psoralen (SSP) to determine the solution structure of a segment of the human beta globin pre-mRNA. In these experiments, SSP is first delivered as monoadducts to specific nucleotides in the pre-mRNA and subsequently used to form intramolecular RNA-RNA cross-links. The use of this reagent greatly decreases the number of the cross-linked products as compared to generalized psoralen cross-linking. The experiments confirm the locations of previously determined aminomethyltrimethylpsoralen (AMT) cross-links in the human precursor mRNA. In addition, new cross-links consistent with an alternative secondary structure and a small number of cross-links that represent higher order interactions have been determined. Altogether, 42 of 47 cross-links identified in this analysis can be accounted for in a small number of alternative secondary structures and higher order interactions. The site directed cross-linking technique will be useful for the precise determination of RNA secondary and tertiary structures under a variety of experimental conditions.
Teare
J
J
Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University Medical Center, MO 63104.
Wollenzien
P
P
D001483:Base Sequence; D003432:Cross-Linking Reagents; D005091:Exons; D011564:Furocoumarins; D005914:Globins / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000235:genetics; D012996:Solutions; D014158:Transcription, Genetic; D014307:Trioxsalen / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012996:Solutions; C035639:2-pyridyldithioethylmethylamidodiethoxyethane methylaminomethyltrimethylpsoralen; D005914:Globins; C018822:aminomethyltrioxsalen; D014307:Trioxsalen