Mutation of glutamate 62 to lysine in yeast transcription factor (TF) IIB (Sua7) causes a cold-sensitive phenotype. This mutant also leads to preferential transcription of downstream start sites on some promoters in vivo. To explore the molecular nature of these phenotypes, the TFIIB E62K mutant was characterized in vitro. The mutant interacts with TATA-binding protein normally. In three different assays, the mutant can also interact with RNA polymerase II and recruit it and the other basal

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... scription factors to a promoter. Despite the ability to assemble a transcription complex, the TFIIB E62K protein is severely defective in transcription in vitro. Therefore, the role of TFIIB must be more than simply bridging TATA-binding protein and polymerase at the promoter. We propose that the region around Glu-62 in yeast TFIIB plays a role in start site selection, perhaps mediating a conformational change in the polymerase or the DNA during the search for initiation sites. This step may be related to the yeastspecific spacing between TATA elements and start sites since mutations of the corresponding glutamate in mammalian TFIIB do not produce a similar effect. Transcription initiation by RNA polymerase II (pol II) 1 in eukaryotes requires an assembly of general transcription factors on the promoter to form a preinitiation complex (PIC) (reviewed in Refs. 1-3). An initial committed complex is formed by TBP/TFIID binding to the TATA element of a promoter (4). Subsequent interaction with TFIIB bridges TFIID on the TATA element and RNA pol II/TFIIF, TFIIE, and TFIIH (5). Several steps occur after transcription complex assembly, including promoter melting, start site selection and initiation, promoter clearance, elongation, and reinitiation. During the initiation process, numerous protein-protein and protein-DNA interactions must be established, adjusted, and then disrupted as the transcription machinery moves away from the start site. The mechanism of transcription start site selection is not well characterized. In higher eukaryotes, promoter melting and transcription initiation overlap at a fixed distance of ϳ25 nucleotides downstream from a TATA box (6, 7), suggesting that start site selection is simply due to the geometry of the transcription complex. In contrast, initiation in Saccharomyces cerevisiae generally occurs at multiple sites within a broad window of 30 -120 nucleotides from TATA (8 -11). Genetic methods have been applied to identify factors that affect start site selection in yeast cells. Mutations in TBP (spt15 alleles (12) ), TFIIB (sua7 (13)), and two polymerase subunits (rpb1/sua8 (14) and rpb9/shi/ssu73 (15-18)) can alter start site selection in vivo. In contrast to the spt mutations, which affect TATA element selection, the sua and shi mutations alter the spacing between the TATA element and initiation sites. Rather than causing completely novel initiation sites to be used, the mutations change the relative usage of normal initiation sites (i.e. strongly favoring upstream or downstream start sites). It is interesting to note that these mutations generally do not affect overall promoter strength and that many promoters are unaffected. In agreement with the genetic experiments, species-specific selection of transcription start sites in S. cerevisiae and Schizosaccharomyces pombe was specified in vitro by pairwise replacement of both TFIIB and pol II (19). TFIIB has two domains that correlate with its two known interactions. At the N terminus is a zinc ribbon domain that is essential for the pol II/TFIIF recruiting activity (20, 21). A proteolytically resistant C-terminal domain of TFIIB is necessary and sufficient for the interaction with the TBP⅐DNA complex (20 -24). The structures of these two domains have been characterized separately (25-27). However, a highly conserved region linking two domains does not appear in the structures and has been proposed to be a flexible "hinge" region. Although the N-terminal domain of TFIIB is absent from the x-ray structure of the DNA⅐TBP⅐TFIIBc complex, this domain is predicted to be close to the initiation site (27) . TFIIB physically links TBP/TFIID and pol II/TFIIF and may thereby define the spacing between them. Some mutations in the TFIIB hinge region can dramatically affect the spacing between the TATA element and the initiation sites in vivo (13, 28, 29) . This phenotype allowed the original isolation of the yeast TFIIB gene (SUA7) as a suppressor of upstream ATG codons (13). Interestingly, these start site selection mutants also exhibit a cold-sensitive phenotype. In an attempt to characterize the role of TFIIB in transcription start site selection, we analyzed one of the Sua7 mutant proteins in vitro. Surprisingly, we found that the Sua7 E62K mutant can assemble transcription complexes normally, but is severely defective in transcription in vitro. These findings indicate that TFIIB is required not only for initial assembly of the transcription complex, but also for a later step in initiation such as promoter melting, start site selection, or promoter clearance. MATERIALS AND METHODS Native Protein Purification-The purification of RNA pol II and TFIIH was described (30). The Mono S (HR 5/5) (Amersham Pharmacia Biotech) fractions obtained during the purification of TFIIH (30) were