The human U1A protein contains three distinct domains: the N-terminal RBD1 (amino acids 1-101), the C-terminal RBD2 (amino acids 195-282), and the linker region (amino acids 102-194). The RBD1 domains of two U1A proteins bind specifically to two internal loops in the 3' untranslated region (3' UTR) of its own pre-mRNA. Tryptophan fluorescence and fluorescence resonance energy transfer data show that the two RBD2 domains do not interact with any regions of the UTR complex and display an overall tumbling that is uncorrelated from the core of the complex (formed by RBD1-UTR), indicating that the linker regions of the two U1A proteins remain flexible. The two RBD2 domains are separated by an apparent distance greater than 74 Å in the UTR complex. The linker region adjacent to the RBD1 domain (103- ERDRKREKRKPKSQETP-119) is supposedly involved in protein-protein interactions (12). A single cysteine, introduced at position 101 or 121 of the U1A protein, was used as a specific attachment site for the fluorophore pair IAEDANS [N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine]/DABMI [4- (dimethylamino)-phenylazophenyl-4'-maleimide]. In the U1A-UTR complex (2:1), the dyes at the 101 position are separated by <R> = ~51 Å, while the dyes at the 121 position are at an apparent distance <R> = ≃58 Å. The 101-121 crossed distance on adjacent U1A proteins averages to <R> = 55 Å. These results suggest that the amino acid sequence 101-121 of the two U1A proteins in the complex are held in proximity to each other in a compact conformation.