Genes For Tfb2, Tfb3, and Tfb4 Subunits of Yeast Transcription/Repair Factor IIH: HOMOLOGY TO HUMAN CYCLIN-DEPENDENT KINASE ACTIVATING KINASE AND IIH SUBUNITS

William John Feaver,N Lynn Henry,Zhigang Wang,Xiaohua Wu,Jesper Q Svejstrup,David A Bushnell,Errol C Friedberg,Roger D Kornberg

doi:10.1074/jbc.272.31.19319

Abstract

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

Highlights

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described
The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36kDa subunit of murine CAK and to the 32-kDa subunit of human TFIIH
Genes for 55- and 38-kDa Subunits of Yeast TFIIH—Hexahistidine-tagged yeast TFIIH was purified to near homogeneity by a procedure involving affinity chromatography on Ni21nitrilotriacetic acid-agarose [2], resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane [29]

Summary

EXPERIMENTAL PROCEDURES

TFIIH Purification and Microsequencing—TFIIH was purified to near homogeneity [2], transferred to polyvinylidene difluoride membrane (Bio-Rad), and stained with Ponceau-S (Sigma) as described [15]. Disruptions of TFB2, TFB3, and TFB4 —To disrupt the TFB2 gene, the AatII (blunt)/NaeI fragment from pRS303 [19] containing the HIS3 gene was cloned between the EcoRV and ClaI (blunt) sites of pBS/TFB2/ Xba/2500/DXB to give pKO/TFB2 By subcloning the approximately 2.5-kb XbaI fragment from pBS/TFB2 containing the entire TFB2 ORF into the XbaI site of Bluescript SK1, followed by removal of the XhoI and BamHI sites in the polylinker by digestion, blunting, and religation. For the disruption of TFB4, a cassette encoding the LEU2 marker was amplified by PCR from pRS305 [19] using primers which contained 17-mer oligonucleotide sequences for the amplification, flanked by 61mer sequences that were homologous to those immediately upstream and downstream from the TFB4 open reading frame, respectively. Strains carrying the correct mutation were sporulated and tetrads were dissected on YPD agar and replica plated onto synthetic media lacking leucine [28]

RESULTS

TABLE II Correlation of yeast and human TFIIH subunits

DISCUSSION