Repression and redirection of Saccharomyces cerevisiae tRNA synthesis from upstream of the transcriptional start site.

Derivatives of the Saccharomyces cerevisiae SUP4 tRNATyr gene with binding sites for the transcription regulatory protein GCN4 located upstream of the transcriptional start site have been constructed. The effect of GCN4 on transcription of these genes by purified RNA polymerase III and transcription factors (TF) IIIB and IIIC has been analyzed. GCN4 effectively blocks initiation of transcription only when prebound to sites that overlap with the binding site of TFIIIB. Residual GCN4-repressed transcription is significantly redirected to nearby downstream sites, the selection of which depends on the location of bound GCN4. That prebound repressing GCN4 redirects, instead of merely blocking, the TFIIIC-dependent interaction of TFIIIB with DNA has been directly demonstrated by footprinting. The effect of GCN4 on transcription persists after it has been stripped off its DNA-binding site: once it has been redirected, DNA-bound TFIIIB remains in place, a consequence of the fact that it binds extraordinarily tightly to DNA without recognizing specific DNA sequence.