蛋白质－DNA相互作用与真核基因转录

结合最近几年对真核转录调节因子和DNA的结构与机能研究,概述了蛋白质-蛋白质及蛋白质-DNA相互作用方式以及介导相互作用的分子结构基础,论述了转录因于之间、转录因子与DNA之间相互作用过程中的协同与拮抗作用、发生机制及其在真核基因转录调节中的普遍性和重要意义.     

真核生物转录因子对DNA序列的识别

真核生物转录因子对ＤＮＡ序列的识别杨岐生（浙江大学生物科学与技术系,杭州３１００２７）关键词真核生物转录因子,蛋白质－ＤＮＡ识别研究蛋白质和ＤＮＡ两类生物大分子的相互作用,以阐明基因表达、调控及信息传递的分子机制,是认识生命活动本质的核心问题。本文介...     

蛋白质磷酸化与基因转录的调节

蛋白质磷酸化与基因转录的调节敖世洲（中国科学院上海生物化学研究所,２０００３１）基因的转录受顺式作用元件和反式作用因子的控制,涉及很多蛋白质与ＤＮＡ,蛋白质与蛋白质之间的复杂反应,在这些反应中,蛋白质的磷酸化与去磷酸化起着重要的调节作用。在已详细研究的转录因子中,都存在磷酸化与非磷酸化的两种形式。磷酸化对转录因子的活性调节主要在三个水平上起作用：控制转录因子从细胞质到细胞核的运转,影响转录因子与特异DNA序列的结合, 调节蛋白质因子转录激活区与其他因子的作用。     

真核基因转录与转录调节相关复合物

真核细胞核中转录因子与染色质模板如何相互作用调节基因转录是基因表达调控研究的一个中心问题.近来的研究表明,参与基因转录的各种调节因子在核内形成多种复合物,如RNA聚合酶Ⅱ全酶、染色质重塑复合物、核小体以及增强小体等.这些复合物之间相互作用,调节染色质结构,在染色质模板上进一步组装成转录复合物,参与转录调节的各个环节,调节转录复合物活性.这些复合物的形成,整合了转录调节的各种信息,提高了转录调节效率,是真核基因有效、严格、有序表达的基础.另一方面,这些复合物的存在给基因表达调控的研究提出了新问题,发展新的研究思路和新的研究技术具有重要意义.     

鸡AchRγ亚基基因远上游增强子样作用依赖生肌因子

先前曾报告鸡ＡＣｈＲγ亚基基因５连接区－５３８／－２８８片段（含一对Ｅ盒子）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用,为进一步观察ＤＮＡ－ｍｙｏｇｅｎｉｎ相互作用对γ基因转录激活功能的影响,利用瞬时表达体系研究了鸡ｎＡＣｈＲγ亚基基因５端－５３８／－２８８片段调控机能,ＣＡＴ分析表明：γ基因－５３８／－２８８片段可显著激活ｔｋ启动子在分化的Ｃ２Ｃ１２肌细胞中的转录活性,但不能激活在未分化Ｃ２Ｃ     

cDNA克隆p14－6肿瘤抑制功能的分子机制

本文探讨了具有肿瘤抑制功能的ｃＤＮＡ克隆Ｐ１４－６（即人白细胞介素６核转录因子ＮＦ－ＩＬ６的３’非翻译区）的ＲＮＡ转录物与回复相关蛋白ＢＮＦ的相互作用,发现该ＲＮＡ与ＢＮＦ的相互作用位点为其３’侧的富Ｕ序列内的１个２４核苷酸片段;并发现ＢＮＦ系一群蛋白质,它们可能先相互结合成１个蛋白质复合物,然后再与ＲＮＡ位点作用．其中可能只有１个蛋白质（Ｒ６２）直接与该ＲＮＡ结合。     

HBVX蛋白与基因调控和肿瘤形成的关系

乙型肝炎病毒Ｘ蛋白是乙型肝炎病毒基因组的四个开读框架之一,Ｘ－ＯＲＦ编码一分子量约为１７ｋＤ,约含有１５４个氨基酸的蛋白质。实验证实ＰＸ是一种反式作用因子,能与细胞内多种与转录、基因调节有关的因子结合,广泛激活病毒和细胞的启动子,参与基因调控,与ＨＢＶ慢性感染后所形成的肝细胞肿瘤密切相关。ＰＸ本身不具有直接结合双链ＤＮＡ的能力,它主要是通过蛋白与蛋白间的相互作用来行使功能,其中包括直接与一般转录因     

真核生物mRNA的帽结构与帽结合蛋白

帽结构是所有ＲＮＡ 聚合酶Ⅱ转录产物的特征性结构,它在ｍ ＲＮＡ 的功能和代谢的很多方面起作用． 在这些过程中还离不开相关蛋白质对它的识别和粘附,作为它行使功能的媒介,这些蛋白质就称为帽结合蛋白（Ｃａｐ－Ｂｉｎｄｉｎｇ Ｐｒｏｔｅｉｎ,ＣＢＰ）． 该文主要讨论了帽结构与胞质中的ＣＢＰ－ｅＩＦ４Ｅ（ｅｕｋａｒｙｏｔｉｃ ｉｎｉｔｉａｔｉｏｎ ｆａｃｔｏｒ ４Ｅ,真核起始因子４Ｅ）的相互作用在ｍ ＲＮＡ 指导的翻译起始中的作用机制,以及帽结构与核内发现的另一种ＣＢＰ复合体相互作用在ｍ ＲＮＡ 加工中的作用．     

转录因子在茄科植物中的研究进展

转录因子是能与真核基因启动子区域特异性相互作用的DNA结合蛋白,通过它们之间或与其他相关蛋白之间的相互作用,能够激活或抑制其转录。茄科(Solanaceae)植物的整个发育进程(营养生长、生殖生长及对于外界环境的响应等)几乎都有转录因子的参与。综述了植物中最主要的几个转录因子家族MYB、NAC、WRKY、MADS、AP2/ERF在茄科植物中的研究进展,以期为茄科植物的研究和利用提供参考。     

反平行β－折叠──蛋白质与DNA结合的新模式

反平行β－折叠──蛋白质与ＤＮＡ结合的新模式史永昶（扬州大学农学院生化教研室,扬州２２５００９）关键词蛋白质－ＤＮＡ结合模式,反平行β－折叠,阻遏蛋白,ＨＵ蛋白蛋白质与ＤＮＡ的相互作用涉及发生在细胞中的许多基本过程,尤其与基因表达和细胞分化密切相关。...     

Pairwise interactions of the six human MCM protein subunits

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p. Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs. The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed. However, the architecture of the human MCM heterohexameric complex is still unknown. We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells. Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex. We also propose models for the structures of subcomplexes. Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.     

Nuclear-mitochondrial epistasis for fitness in Saccharomyces cerevisiae

In addition to the familiar possibility of epistasis between nuclear loci, interactions may evolve between the mitochondrial and nuclear genomes in eukaryotic cells. We looked for such interactions in Saccharomyces cerevisiae genotypes evolved independently and asexually in the laboratory for 2000 generations, and in an ecologically distinct pathogenic S. cerevisiae strain. From these strains we constructed derivatives entirely lacking mitochondrial DNA and then used crosses to construct matched and unmatched pairings of nuclear and mitochondrial genomes. We detected fitness effects of such interactions in an evolved laboratory strain and in crosses between the laboratory and pathogen strains. In both cases, there were significant contributions to progeny fitness of both nuclear and mitochondrial genomes and of their interaction. A second evolved genotype showed incompatibility with the first evolved genotype, but the nuclear and mitochondrial contributions to this incompatibility could not be resolved. These results indicate that cytonuclear interactions analogous to those already known from plants and animals can evolve rapidly on an evolutionary timescale.     

The eukaryotic replication complex and its affinity modification analysis

Replication of eukaryotic DNA is driven by a protein complex, in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases alpha, delta, and epsilon involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is in an interplay with DNA replication. The function of the entire multicomponent system is regulated by protein--nucleic acid and protein--protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

染色质互作相关转录因子的挖掘及功能分析

染色质互作是真核生物基因组组装的基础,并且在调控真核基因细胞特异性表达中发挥重要作用.染色质互作的发生与特定的蛋白质有关,目前已经发现CTCF (CCCTC binding facor,转录阻抑物)和黏连蛋白与染色质互作相关,然而并不清楚是否还有其他蛋白质参与染色质互作.我们将整合高通量染色体构象捕获(Hi-C)和染色质免疫沉淀-测序(ChIP-seq)数据,在GM12878和K562细胞系中挖掘与染色质互作相关的转录因子,并对发现的转录因子做功能分析.我们在频繁发生互作的染色质位点中发现RUNX3、SPI1等转录因子也可能参与染色质互作.另外,通过FP-growth的数据挖掘方法还发现多个转录因子可能协同作用参与染色质互作.研究结果将为染色质互作相关实验的开展提供先验知识.     

A domain model for eukaryotic DNA organization: a molecular basis for cell differentiation and chromosome evolution

A model for eukaryotic chromatin organization is presented in which the basic structural and functional unit is the DNA domain. This simple model predicts that both chromosome replication and cell type-specific control of gene expression depend on a combination of stable and dynamic DNA-nuclear matrix interactions. The model suggests that in eukaryotes, DNA regulatory processes are controlled mainly by the intranuclear compartmentalization of the specific DNA sequences, and that control of gene expression involves multiple steps of specific DNA-nuclear matrix interactions. Predictions of the model are tested using available biochemical, molecular and cell biological data. In addition, the domain model is discussed as a simple molecular mechanism to explain cell differentiation in multi-cellular organisms and to explain the evolution of eukaryotic genomes consisting mainly of repetitive sequences and "junk" DNA.     

Insight into initiator-DNA interactions: a lesson from the archaeal ORC

Although initiation of DNA replication is considered to be highly coordinated through multiple protein-DNA and protein-protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences. Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins.