EQUIVALENT MUTATIONS IN THE 2 REPEATS OF YEAST TATA-BINDING PROTEIN CONFER DISTINCT TATA RECOGNITION SPECIFICITIES

To investigate the process of TATA box recognition by the TATA box-bin ding protein (TBP), we have performed a detailed genetic and biochemic al analysis of two Saccharomyces cerevisiae TBP mutants with altered D NA-binding specificity. The mutant proteins have amino acid substituti ons (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mu tations of the consensus TATAAA sequence, one of the TBP mutants (TBP- L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP -L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box . Specificity patterns determined from in vitro transcription experime nts with purified recombinant wild-type TBP and TBP-L205F agree closel y with those observed in vivo, indicating that altered TATA utilizatio n in the mutant strains is a direct consequence of altered TATA recogn ition by the mutant TBPs. The distinct TATA recognition patterns exhib ited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP bin ds to the TATA element in a specific orientation. The orientation pred icted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This ori entation is. consistent with that observed in vitro by crystallographi c analyses of TBP-TATA box complexes. Finally, the importance of alter ed DNA-binding specificity in transcriptional regulation at the S. cer evisiae his4-912 delta promoter was addressed for TBP-L205F. A mutatio nal analysis of this promoter region demonstrates that the nonconsensu s TATA element CATAAA is required for a transcriptional effect of TBP- L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in tr anscription initiation.