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

David L. Beck, W. Thomas Stump, Kathleen B. Hall

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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.

Original languageEnglish
Pages (from-to)331-339
Number of pages9
Issue number3
StatePublished - Mar 1998


  • Hydroxyl radical cleavage
  • RNA-protein complexes
  • U1A RBD1-EDTA(Fe)
  • UTR


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