Analysis by Engineering for Two DNA Binding Proteins That Regulate The Initiation of Ribosomal RNA Synthesis.

对调节核糖体 RNA 合成起始的两种 DNA 结合蛋白进行工程分析。

• 批准号: 04670137
• 负责人: NISHIMURA Tetsuji
• 金额: $ 1.34万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670137/
• 关键词: Ribosomal RNA Gene; DNA Binding Proteins; Promoter Region; CAT Assay; Transcriptional Regulation; In Vitro rRNA Synthesis; House-keeping Gene; Genetic Engineering; 核移行シグナル; 核小体; HMG‐box

It is necessary two DNA binding proteins, Upstream Binding Protein(UBF)and SL-1, for formation of the initiation complex of ribosomal RNA gene(rDNA)transcription. Mouse UBF has a nuclear localization sequence in the 4th HMG-box. Deletion of either the HMG-box1, a region crucial for rDNA binding, or the acidic tail, a region that may interasct with SL-1, results in the loss of necleolar targeting. HMG-box1 is necessary for UBF to bind to the upstream control element of the rDNA.Mouse UBF is transferred to the nucleus by its NLS and is sequestered in the nucleous by its specific and stable binding to the rDNA promoter via HMG-boxes and the acidic tail.The region between the transcriptional initiation site and -1200nt shows the promoter activities. In this region, there are several GC-boxes and three kinds of serum response elements, but no TATA-box or CAT-box. The existence of those cis acting element is necessary for regulation of the UBF gene. The mRNA of UBF was expressed 4 times after nutritional shift-up. These observation indicates UBF gene is one of the house-keeping genes and is regulated through several kinds of element in the promoter region correspondingly with growth-stimulation signals. SL-1 is constituted with 5 kinds of proteins, containing TATA binding protein(TBP), at least. UBF binds with protein associated TBP.Ribosomal RNA synthesis in vitro is inhibited with anti-body against TBP.So TBP binds the promoter region of rDNA.And then UBF makes a stable complex with SL-1/DNA complex as pre-initiation complex of rDNA transcription.

核糖体RNA基因(RDNA)转录起始复合体的形成需要两个DNA结合蛋白：上游结合蛋白(UBF)和SL-1。小鼠UBF在第4个HMG盒中有核定位序列。缺失HMG-box1(对rDNA结合至关重要的区域)或酸性尾部(可能与SL-1相互作用的区域)都会导致核仁靶向性的丧失。HMG-box1是UBF与rDNA上游调控元件结合所必需的。小鼠UBF通过其NLS转移到细胞核，并通过HMG-box和酸性尾巴与rDNA启动子特异而稳定地结合在核中。转录起始点与-1200nt之间的区域显示了启动子的活性。该区域有多个GC盒和三种血清反应元件，但没有TATA盒和CAT盒。这些顺式作用元件的存在是UBF基因调控所必需的。UBF基因在营养上移后表达了4次。这些观察表明，UBF基因是一个看家基因，受启动子区域的几种元件的调控，相应地有生长刺激信号。SL-1由5种蛋白质组成，至少含有TATA结合蛋白(TBP)。UBF与蛋白质结合，体外核糖体RNA合成被抗TBP抗体抑制，TBP与rDNA启动子区域结合，UBF与SL-1/DNA形成稳定的复合体，作为rDNA转录的预启动复合体。

项目成果

Yukio Mishima,Tetsuji Nishimura,Masami Muramatsu,Ryo Komirami.: "Transcription of Mouse Ribosomal RNA Gene with Inactive Extracts Is Activated by NAD^+ In Vitro." J.Biochem.113. 36-42 (1993)

Yukio Mishima、Tetsuji Nishimura、Masami Muramatsu、Ryo Komirami.：“非活性提取物的小鼠核糖体 RNA 基因的转录在体外被 NAD^ 激活。”

Y.Maeda et al.: "Mouse rRNA gene transcription factor mUBF requires both HMG-box1 and an acidic tail for nucleolar accumulation : Molecular analysis of the nucleolar targeting mechanism." EMBO J.11. 3695-3704 (1992)

Y.Maeda 等人：“小鼠 rRNA 基因转录因子 mUBF 需要 HMG-box1 和酸性尾部来进行核仁积累：核仁靶向机制的分子分析。”

Yasushi Maeda: "Mouse rRNA gene transcription factor mUBF reguires both HMG-bonland an acidictail for nucleolar aceumulation:molecular analysis of the nucleolar targeting nuoleclassigus." The EMBO Journal. 11. 3695-3704 (1992)

Yasushi Maeda：“小鼠 rRNA 基因转录因子 mUBF 需要 HMG-bonland 和酸尾进行核仁积聚：核仁靶向 nuoleclassigus 的分子分析。”

Y.Maeda et al.: "Mouse rRNA gene transcription factor mUBF requires both HMG-boxland an acidic tail for nucleolar accumulation:molecular analysis of the nucleolar targeting mechanism" EMBO J.11. 3695-3704 (1992)

Y.Maeda 等人：“小鼠 rRNA 基因转录因子 mUBF 需要 HMG-box 和酸性尾部进行核仁积累：核仁靶向机制的分子分析”EMBO J.11。

Yukio Mishima: "Transcription of Mouse Ribosomal RNA Gene with Inactive Extracts Is Activated by NAD^+ In Vitro" J.Biochemistry. 113. 36-42 (1993)

Yukio Mishima：“非活性提取物的小鼠核糖体 RNA 基因的转录在体外被 NAD^ 激活”J.Biochemistry。