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Defining the orientation of the human U1A RBD1 on its UTR by tethered-EDTA(Fe) cleavage

Published online by Cambridge University Press:  01 March 1998

DAVID L. BECK ,
W. THOMAS STUMP  and
KATHLEEN B. HALL
DAVID L. BECK
Affiliation:
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
W. THOMAS STUMP
Affiliation:
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
KATHLEEN B. HALL
Affiliation:
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
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Abstract

The N-terminal RNA binding domain of the human U1A protein (RBD1) specifically binds an RNA hairpin of U1 snRNA as well as two internal loops in the 3′ UTR of its own mRNA. Here, a single cysteine has been introduced into Loop 1 of RBD1, which is subsequently used to attach (EDTA-2-aminoethyl) 2-pyridyl disulfide-Fe3+ (EPD-Fe). This EDTA-Fe derivative is used to generate hydroxyl radicals to cleave the proximal RNA sugar–phosphate backbone in the RNA–RBD complexes. RBD1(K20C)–EPD-Fe cleaves the 5′ strand of the RNA hairpin stem, centered four base pairs away from the base of the loop, and cleaves the UTR in two places, again centered on the 5′ side of the fourth base pair from each internal loop. These data, extrapolated to the position of Lys 20 in RBD1, orient the two proteins bound to the UTR, and provide direct biochemical evidence for the proposed model of the RBD1:UTR complex.

Keywords

hydroxyl radical cleavageRNA–protein complexesU1A RBD1–EDTA(Fe)UTR
Type
Research Article
Information
RNA , Volume 4 , Issue 3 , March 1998 , pp. 331 - 339
Copyright
© 1998 RNA Society

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