Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C.

A physical DNA binding assay was employed to analyze the binding of yeast RNA polymerase III transcription factor C (TFIIIC) to tRNA genes. The assay allowed us to measure the equilibrium constants for specific and nonspecific TFIIIC-DNA binding and to assess the effects of tRNATyr-DNA gene promoter mutations on binding. Sequence alterations in the B block element of the promoter greatly affect the equilibrium constant for specific TFIIIC binding (K8). Mutations which decrease tRNATyr homology to the recognized B block consensus sequence drastically reduce K8 (43- to 370-fold), while mutations which increase homology increase K8 (4- to 5-fold). By contrast, point mutations in the A block element of the tRNATyr promoter have less than 2-fold effects on K8; however, total deletion of A block sequences reduces K8 2- to 5-fold. These results indicate that TFIIIC-rTNA gene binding involves interactions with both A block and B block sequences, but B block interactions dominate, and the relative contribution of A block interactions is small. Since A block sequences are absolutely required for active tRNA gene transcription, the binding results suggest that the role of A block sequences in transcription is not confined solely to TFIIIC binding.