CROSS-LINKING OF AN IODO-URIDINE-RNA HAIRPIN TO A SINGLE-SITE ON THE HUMAN U1A N-TERMINAL RNA-BINDING DOMAIN

The N-terminal RNA binding domain (RED) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-n ucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in tu rn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking res ults described here, and in more detail by a recently described co-cry stal of this RED with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking exper iments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contr ast to the wildtype sequence, which does not. This single uridine subs titution in the 10-nucleotide loop is the site of crosslinking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RED. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.