The N-degron strategy has been used to generate a yeast strain harboring a temperature-sensitive allele of TFB4 (tfb4 td ), the gene that encodes the 37-kDa subunit of the transcription/repair factor TFIIH. The tfb4 td strain was sensitive to UV radiation and is defective in nucleotide excision repair in vitro. The mutant strain was also found to be an inositol auxotroph due at least in part to an inability to properly induce expression of the INO1 gene. These results indicate that like other subunits of TFIIH, Tfb4 is required for both RNA polymerase II transcription and DNA repair.
The holo transcription factor IIH (TFIIH)1 is comprised of nine subunits and has been shown to be required for both transcription by RNA polymerase II (RNAP II) and nucleotide excision repair (NER) (1, 2). The recent identification of the TFB2, TFB3, and TFB4 genes encoding the 55-, 38-, and 37-kDa subunits, respectively, completed the molecular definition of this yeast transcription factor (2). All yeast IIH subunits are encoded by essential genes, consistent with an essential role in transcription. In addition each yeast subunit has a highly conserved counterpart in human TFIIH (2).The NER pathway is required for the repair by excision of a myriad of helix-distorting lesions, although it is perhaps best characterized with respect to its ability to remove 6 -4 photoproducts and cyclobutane pyrimidine dimers, resulting from exposure to UV radiation (3). Following base damage recognition, DNA surrounding lesions is locally unwound and incised at double-strand/single-strand junctions by two junction-specific endonucleases with opposite strand polarity. Damaged bases are then excised as short, single-stranded oligonucleotides. Repair synthesis and ligation complete the process of NER. In yeast, localized unwinding during NER is catalyzed by the Rad3 and Ssl2 DNA helicases, both subunits of TFIIH (1). Similarly, promoter melting during initiation by RNAP II is thought to require the activity of Ssl2 (4). It seems likely that the common requirement for localized unwinding is the functional basis for TFIIH involvement in both NER and RNAP II transcription.To date viable and conditional mutations in the yeast IIH subunits Ssl2, Rad3, Tfb1, Tfb2, Ssl1, and TFB3 have been utilized to demonstrate a direct requirement for these subunits in NER (2, 5, 6).2 Holo TFIIH can be further divided into core TFIIH, comprising the seven subunits described above, and the subcomplex TFIIK (8). TFIIK, comprised of the Ccl1 and Kin28 proteins, has protein kinase activity directed toward the Cterminal domain of Rpb1, the largest subunit of RNAP II (9 -11). No role for TFIIK in NER has been demonstrated.In this study we have used the N-degron approach of Varshavsky and colleagues (12) to generate a strain carrying a temperature-sensitive allele of TFB4. Characterization of this strain revealed UV radiation sensitivity, defective NER in vitro, and impaired induction of INO1, leading to inositol auxotrophy. Based on these results we conclude that Tfb4 is require...