REGIONAL CODON RANDOMIZATION - DEFINING A TATA-BINDING PROTEIN SURFACE REQUIRED FOR RNA POLYMERASE-III TRANSCRIPTION

The TATA-binding protein (TBP) is required for transcription by all th ree nuclear RNA polymerases. TBP was subjected to regional codon rando mization, a codon-based mutagenesis method that generates complex yet compact protein libraries. Analysis of 186 temperature-sensitive TBP m utants yielded 65 specifically defective in transcription by RNA polym erase III (Pol III). These mutants map to a limited TBP surface that m ay interact with Tds4, a component of the Pol III transcription factor TFIIIB. Strains that contain the Pol III-defective derivatives have i ncreased amounts of messenger RNA, which suggests that competition amo ng TBP-interacting factors for limiting quantities of TBP determines t he ratio of Pol II and Pol III transcription in vivo.