To see if the domains of Sbp1 bind to the 5′UTRPab1 cooperatively, we studied binding properties of individual domains of Sbp1 to 5′UTRPab1 and compared the results with the wild-type Sbp1. Care was taken to ensure that individual domains—RRM1, RGG, and RRM2 domains of Sbp1 (Sbp1RRM1, Sbp1RGG, and Sbp1RRM2)—were folded properly (Supplemental Fig. S2B). As shown in Figure 1D, while individual domains of Sbp1 bind to the 5′UTRPab1, they interact with the RNA with much weaker affinities compared to the affinity of the full-length Sbp1 with the 5′UTRPab1. The measured apparent dissociation constant (Kd) of full-length Sbp1 to the 5′UTRPab1 is 26.5 ± 1.1 nM with a Hill coefficient of 2.1 ± 0.1 (Supplemental Fig. S3A). In contrast, under the same experimental conditions, the Kd values of its domain mutants, the Sbp1RRM1, Sbp1RRM2, and Sbp1RGG to the 5′UTRPab1 are about 1.1 μM, 0.9 μM, and 2.7 μM, respectively (data not shown). Thus, the binding of full-length Sbp1 to 5′UTRPab1 is over 40 times stronger than that of its individual domains to the same RNA. These results demonstrate convincingly that the domains of Sbp1 bind to the 5′UTRPab1 cooperatively.