Chew Boon Shang, Lehming Norbert
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore.
Biochem J. 2007 Sep 1;406(2):265-71. doi: 10.1042/BJ20070441.
The TBP (TATA-box-binding protein), Tbp1p, plays a vital role in all three classes of transcription by RNA polymerases I-III. A TBP1(E186D) mutation had been described that affected interaction of Tbp1p with TFIIB (transcription factor IIB) and that caused slow-growth, temperature-sensitivity, 3-aminotriazole-sensitivity as well as a gal(-) phenotype. We used the TBP1(E186D) mutant for suppressor screens, and we isolated TFIIB/SUA7(E202G) as an allele-specific suppressor of all phenotypes caused by the TBP1(E186D) mutation. Our results show that the SUA7(E202G) mutation restored binding of TFIIB to Tbp1(E186D)p. In addition, we observed that Tbp1(E186D)p was expressed at a lower level than wild-type Tbp1p, and that SUA7(E202G) restored the protein level of Tbp1(E186D)p. This suggested that the TBP1(E186D) mutation might have generated its phenotypes by making Tbp1p the limiting factor for activated transcription. DNA microarray analysis indicated that the TBP1(E186D) temperature-sensitivity and slow-growth phenotypes might have been caused by insufficient amounts of Tbp1p for efficient transcription of the rRNA genes by RNA polymerase I.
TBP（TATA盒结合蛋白），即Tbp1p，在RNA聚合酶I - III的所有三类转录过程中都起着至关重要的作用。此前已报道过一种TBP1（E186D）突变，该突变影响Tbp1p与TFIIB（转录因子IIB）的相互作用，并导致生长缓慢、温度敏感性、3 - 氨基三唑敏感性以及gal（-）表型。我们使用TBP1（E186D）突变体进行抑制子筛选，并分离出TFIIB/SUA7（E202G）作为TBP1（E186D）突变所导致的所有表型的等位基因特异性抑制子。我们的结果表明，SUA7（E202G）突变恢复了TFIIB与Tbp1（E186D）p的结合。此外，我们观察到Tbp1（E186D）p的表达水平低于野生型Tbp1p，并且SUA7（E202G）恢复了Tbp1（E186D）p的蛋白水平。这表明TBP1（E186D）突变可能通过使Tbp1p成为激活转录的限制因素而产生其表型。DNA微阵列分析表明，TBP1（E186D）的温度敏感性和生长缓慢表型可能是由于Tbp1p的量不足，无法使RNA聚合酶I有效地转录rRNA基因所致。
