CONTRIBUTION OF INDIVIDUAL BASE-PAIRS TO THE INTERACTION OF TFIIIA WITH THE XENOPUS 5S RNA GENE

The effects of a series of point mutations within the Xenopus borealis somatic-type 5S RNA gene on transcription factor IIIA (TFIIIA) bindin g affinity were quantified. These data define a critical sequence-depe ndent contact region within the classical box C promoter element from base pair 80 to 91. Substitution of GC base pairs at positions 81, 85, 86, 89, and 91 significantly reduce TFIIIA binding affinity. Base pai rs located at other positions within the box C contact region provide a moderate contribution to TFIIIA-5S gene interaction. In contrast to the extensive set of sequence contacts within the box C element, TFIII A interaction is localized primarily to two GC base pairs at positions 70 and 71 within the intermediate promoter element. A selected amplif ication and binding assay (SAAB) was performed with a synthetic intern al control region (ICR) randomized from base pair 78 to 95 to identify box C promoter sequences bound with high affinity by TFIIIA. The wild -type 5S RNA gene sequence from 79 to 92 is strongly selected. These r esults are consistent with the critical role of the box C element in s equence-dependent promoter recognition by TFIIIA.