Nakadai Tomoyoshi, Shimada Miho, Shima Daisuke, Handa Hiroshi, Tamura Taka-Aki
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan.
J Biol Chem. 2004 Feb 27;279(9):7447-55. doi: 10.1074/jbc.M305412200. Epub 2003 Oct 21.
TBP-like protein (TLP) is structurally similar to the TATA-binding protein (TBP) and is thought to have a transcriptional regulation function. Although TLP has been found to form a complex with transcription factor IIA (TFIIA), the in vivo functions of TFIIA for TLP are not clear. In this study, we analyzed the interaction between TLP and TFIIA. We determined the biophysical properties for the interaction of TLP with TFIIA. Dissociation constants of TFIIA versus TLP and TFIIA versus TBP were 1.5 and 10 nm, respectively. Moreover, the dissociation rate constant of TLP and TFIIA (1.2 x 10(-4)/m.s was significantly lower than that of TBP (2.1 x 10(-3)/m.s). These results indicate that TLP has a higher affinity to TFIIA than does TBP and that the TLP-TFIIA complex is much more stable than is the TBP-TFIIA complex. We found that TLP forms a dimer and a trimer and that these multimerizations are inhibited by TFIIA. Moreover, TLP mutimers were more stable than a TBP dimer. We determined the amounts of TLPs in the nucleus and cytoplasm of NIH3T3 cells and found that the molecular number of TLP in the nucleus was only 4% of that in the cytoplasm. Immunostaining of cells also revealed cytoplasmic localization of TLP. We established cells that stably express mutant TLP lacking TFIIA binding ability and identified the amino acids of TLP required for TFIIA binding (Ala-32, Leu-33, Asn-37, Arg-52, Lys-53, Lys-78, and Arg-86). Interestingly, the level of TFIIA binding defective mutant TLPs in the nucleus was much higher than that of the wild-type TLP and TFIIA-interactable mutant TLPs. Immunostaining analyses showed consistent results. These results suggest that the TFIIA binding ability of TLP is required for characteristic cytoplasmic localization of TLP. TFIIA may regulate the intracellular molecular state and the function of TLP through its property of binding to TLP.
TBP样蛋白（TLP）在结构上与TATA结合蛋白（TBP）相似，被认为具有转录调控功能。尽管已发现TLP与转录因子IIA（TFIIA）形成复合物，但TFIIA在体内对TLP的功能尚不清楚。在本研究中，我们分析了TLP与TFIIA之间的相互作用。我们确定了TLP与TFIIA相互作用的生物物理特性。TFIIA与TLP以及TFIIA与TBP的解离常数分别为1.5和10 nM。此外，TLP与TFIIA的解离速率常数（1.2×10⁻⁴/s）显著低于TBP（2.1×10⁻³/s）。这些结果表明，TLP对TFIIA的亲和力高于TBP，并且TLP-TFIIA复合物比TBP-TFIIA复合物稳定得多。我们发现TLP形成二聚体和三聚体，并且这些多聚化受到TFIIA的抑制。此外，TLP多聚体比TBP二聚体更稳定。我们测定了NIH3T3细胞核和细胞质中TLP的含量，发现细胞核中TLP的分子数仅为细胞质中的4%。细胞免疫染色也显示TLP定位于细胞质。我们建立了稳定表达缺乏TFIIA结合能力的突变型TLP的细胞，并确定了TFIIA结合所需的TLP氨基酸（Ala-32、Leu-33、Asn-37、Arg-52、Lys-53、Lys-78和Arg-86）。有趣的是，细胞核中TFIIA结合缺陷型突变TLP的水平远高于野生型TLP和可与TFIIA相互作用的突变TLP。免疫染色分析显示了一致的结果。这些结果表明，TLP的TFIIA结合能力是TLP特征性细胞质定位所必需的。TFIIA可能通过其与TLP结合的特性来调节TLP的细胞内分子状态和功能。
