植物中的Dof 蛋白和Dof转录因子家族

Dof(DNA binding with one finger)蛋白是植物所特有的一类转录因子.其N末端保守的单锌指Dof结构域是既与DNA又和蛋白相互作用的双重功能域,识别的核心序列是AAAG;其C末端氨基酸序列较为多变,是Dof蛋白的特异转录调控结构域.Dof类转录因子在植物中发挥着多种功能.拟南芥和水稻的Dof转录因子家族为Aa、Bb、Cc、Dd 4个同源基因群,同类群间可能具有相似或相反的功能.文章就这一问题的研究进展作了介绍.     

POU蛋白调节中枢神经系统发育

POU蛋白是一组DNA特异的转录调节因子,属同源异形序列超家族.发育过程中,POU蛋白编码基因在中枢神经系统各部位的时空性表达决定神经细胞的发育与分化.     

POU同源域蛋白的结构及发育中的功能

POU同源域蛋白含有两个保守的亚结构域以及它们之间有变化的连接区.两个亚结构域与DNA相互作用,在连接区可塑性和辅因子的帮助下,POU蛋白作为调控因子和转录因子,显示出错综复杂的DNA结合和识别能力.在脊椎动物和无脊椎动物中,POU蛋白参与胚胎发生的早期过程,在细胞谱系的分化和神经发育中起调控作用.     

同源异形基因浅说

同源异形基因是决定果蝇体节形成的发育基因,其同源异形盒子编码同源异形结构域蛋白,在进化上极为保守,从低等到高等动物的基因组中都有存在。其表达具严格的时、空特异性,可控制细胞的分化状态及表型,推测可能是通过对其他基因的调节而发挥作用。最近表明线虫及哺乳类细胞中的转录因子也具有同源异形蛋白,初步证明它们也是转录因子,这对解释同源异形蛋白的功能、探索基因表达的调节机制有重要意义。     

同源异形盒基因和肿瘤

最近,美国哈佛药学院的Ｆｏｒｄ发现同源异形盒(ｈｏｍｅｏｂｏｘ)基因在细胞周期、发育以及癌症发展中起着重要的链接作用[1]同源异形盒基因是与细胞正常分化、发育相关的转录因子,最初作为果蝇同源异形突变中的重要座位而得名。此后,陆续有170多个同源异形盒基因在各类物种中被发现。同源异形盒基因的共同点是具有183个核苷酸长度的同源区,编码61个氨基酸。同源异形盒基因同源区表达蛋白通过其ＤＮＡ结合活性及其下游基因转录活性发挥作用。Ｌａｗｒｅｎｃｅ等[2]推测哺乳动物同源异形盒表达蛋白的作用目标为编码细胞外层蛋白质的基因,粘…     

E2F是疾病治疗的潜在靶点

竞争性寡聚脱氧核糖核酸识别转录因子的DNA结合域,抑制了转录因子对基因的转录调控,转录因子E2F作为潜在的治疗靶点,可以用于抑制肾小球系膜细胞和血管内皮细胞的增生,在部分异常增生的疾病中显示了一定的应用前景。     

一种特异识别SV40启动子的人工转录因子的构建

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合域与效应域两部分组成,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术,筛选到与SV40启动子上9bp序列特异结合的锌指结构,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV40下游的报告基因的表达有很显著的抑制作用。     

同源盒基因(Hox)与哺乳动物生殖

哺乳动物的同源盒基因（Hox）与果蝇的同源异形基因是同源基因,该基因编码的DNA片段含183碱基对,转录由61个氨基酸残基组成的蛋白质保守结构域,称同源异型域.Hox基因碱基顺序及在染色体中的位置都是高度保守的.Hox基因在体节结构分化等空间信息调控中起着重要作用,按特异的空间模式赋予每一体节其自身的特点.近年来的研究表明,Hox基因不但影响胚胎发育,而且与成体生殖系统分化有关,在着床期子宫接受态的建立及子宫蜕膜反应的发生等生殖过程中起着重要的调节作用.     

同源域的结构与功能

在过去的十年中,有关同源框（Homeobox)基因的研究进展异常迅速。同源框基因是控制动物胚胎发育的一类主控制基因。它们都含有一个由180个核苷酸组成的同源框序列;该序列编码的酸区域被称为同源域（Homeodomain)。同源框与同源域在所有后生动物中都高度保守。由于同源域涉及蛋白对DNA中特定核苷酸序列的识别与结合,对其结构与功能的研究有着重要的理论意义。本文综述了有关一些最新进展,包括同源域的三维结构、对DNA的识别模型、与DNA结合的特异性、其功能特异性、Antp同源域的跨膜转动及其在神经元分化中的作用等。（一）经过高分辨率的核磁共振技术和晶体X－射线衍射发现：它的结构包括螺旋I、螺旋I与II之间的连接环、螺旋II、转折区、螺旋Ⅲ、螺旋IV和呈无规则卷曲状态的两个末端;其中、螺旋I与Ⅱ之间,以6个氨基酸相连呈反向平等状态,螺纹旋Ⅲ、IV则与前两个螺旋垂直排列,而螺旋II与III则又紧密连接成一个螺旋－转折－螺旋的结构。另外,同源域的三维结构是同由一个疏水内核聚拢起来的。（二）经过核磁共振研究,建立了Antp同源域单体与14个碱基对DNA双螺旋之间结合的模型。该模型显示：Antp的识别螺旋（螺旋III与IV）在DNA双螺旋的大沟与DNA中ATTA序列的5′端结合,其N端的“臂”在双螺旋的小沟与ATTA序列的3′端结合,位于螺旋I与II之间的氨基酸环则在大沟另一侧与DNA的双螺旋骨架部分互相作用。（三）与同源域多肽相结合的DNA序列中大都含有一个四苷酸序列ATTA（在互补链上为TAAT）的核心结构花式序列,该序列能被不同的同源域蛋白有选择地结合,在该序列之前还有一个二核苷酸序列,可被不同的基酸残然识别。同源域这种结构特性决定了其功能特异性。这可能是以两种匠有机结合来决定的：一方面,不同的同源域蛋白在不同的辅助因子协助下共同完成对DNA的特异识别,另一方面,不同的同源域蛋白识别位点可以直接发挥作用。（四）值得注意的是,Antp同源域还能穿过神经元的细胞膜,进入到细胞核中,该过程不依赖于能量并且加速了神经元的形态分化。其机制还是一个迷。在LIM类和Hox类同源域中也观察到类似现象;很有可能,同源域在控制细胞分化的过程中起着关键作用。     

转录因子与DNA识别的立体化学规律

转录因子-DNA的识别包含普遍性的化学规律和特异性的立体化学规律.转录因子的识别螺旋以特异性方式结合于DNA,位于一侧的大沟内.识别螺旋的“残基连线”和碱基的“碱基连线”之间吻合使两者完全匹配,它通常涉及最多3圈α螺旋和3～5 bp.决定氨基酸残基和碱基相互关系的结合几何图形表示为立体化学图,它表明了识别的特异性.在该基础上总结成转录因子识别DNA的立体化学规律.     

Cooperative DNA‐binding and sequence‐recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al–Cll–DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence‐recognition mechanism of DNA by direct interactions. The residues in the extended N‐terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C‐terminus of the homeodomain interacts with Al to stabilize and localize the third α helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.     

Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1-Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three-amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional alpha helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.     

Conformational changes in the PBX homeodomain and C-terminal extension upon binding DNA and HOX-derived YPWM peptides

PBX is a member of the three amino acid loop extension (TALE) class of homeodomains. PBX binds DNA cooperatively with HOX homeodomain proteins that contain a conserved YPWM motif. The amino acids immediately C-terminal to the PBX homeodomain increase the affinity of the homeodomain for its DNA site and HOX proteins. We have determined the structure of the free PBX homeodomain using NMR spectroscopy. Both the PBX homeodomain and the extended PBX homeodomain make identical contacts with a 5'-TGAT-3' DNA site and a YPWM peptide. A fourth alpha-helix, which forms upon binding to DNA, stabilizes the extended PBX structure. Variations in DNA sequence selectivity of heterodimeric PBX-HOX complexes depend on the HOX partner; however, a comparison of five different HOX-derived YPWM peptides showed that each bound to PBX in the same way, differing only in the strength of the association.     

Isolation and sequence-specific DNA binding of the Antennapedia homeodomain.

The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was overproduced in a T7 expression vector in Escherichia coli. The corresponding polypeptide of 68 amino acids was purified to homogeneity. The homeodomain was analysed by ultracentrifugation and assayed for DNA binding. The secondary structure of the isolated homeodomain was determined by nuclear magnetic resonance spectroscopy. DNA-binding studies indicate that the isolated homeodomain binds to DNA in vitro. It selectively binds to the same sites as a longer Antp polypeptide and a full-length fushi tarazu (ftz) protein. Therefore, the homeodomain represents the DNA-binding domain of the homeotic proteins.     

Characterization of the bacterially expressed Drosophila engrailed homeodomain.

To investigate the mechanism of DNA recognition by the homeodomain, truncated proteins containing the entire homeodomain encoded by the Drosophila engrailed gene were expressed in Escherichia coli. Each protein was accumulated to an amount representing more than 40% of the total bacterial protein and recovered in the soluble fraction. Of the three truncated proteins, the shortest one (71 amino acid residues) was further purified by conventional chromatography. The purified engrailed homeodomain (En-HD) protected a DNA sequence, TTAATT, the core element of consensus sequences recognized by many other homeodomain proteins, from DNase I digestion. UV-CD spectra of the En-HD showed that it mainly consisted of alpha-helix. Based on one-dimensional 1H-NMR spectra, the tertiary structure of the En-HD was shown to be stable against temperature up to 50 degrees C and low pH. The low pH resistance of the protein was also demonstrated by UV-CD measurement. Thus, the current over-production system provides an active and stable homeodomain, which is suitable for structure-function analysis.