To corroborate the UV-cross-link results, filter-binding assays were performed with increasing amounts of Hax-1 protein and a constant amount of the same test RNAs (hairpin-containing region-H, antisense-A and mutant-Hmut). Data were analyzed on a Klotz plot ( Figure 8 ) and the apparent K d for each complex was calculated. The fit of a nonlinear binding curve to the experimental data points was best when calculations were performed in respect to dimer concentration, which indicates that the RNA interacts only with a dimeric form of the protein, thus confirming the UV-cross-link results. The approximate K ddim established from the titrated glutaraldehyde cross-linking experiments (13.5 mM AE 5.5) was in the same range as the K ddim predicted by curve-fitting (fitting performed simultaneously for the H and Hmut data point series in respect to variable K d and constant K ddim values yielded a K ddim of 10 mM AE 4). Transcript H showed the greatest affinity for Hax-1, with a K d of 28 nM AE 7, and significantly lower Table 1 . Three-hybrid screen of a rat testis cDNA library proteins interacting with the M2 hairpin-containing region of the Pol b mRNA 3 0 UTR Plasmids used for transformation of R40C-W Growth on 25 mM 3-AT b-gal plate test