在同源二聚体转录因子识别螺旋相对运动中发现平面摆动现象

目的:考察同源二聚体转录因子的2个识别螺旋在自由状态下和结合状态下相对运动的特征。方法:从蛋白质结构数据库(PDB)得到1R4I、3JXB、1KB2、1LE8等4个复合物结构文件,分别在自由状态和结合状态下用NAMD进行16 ns分子动力学模拟,将2个识别螺旋所成角分解为垂直偏移角和水平偏移角,并做散点图。结果:2个中心对称结合的同源二聚体转录因子在2种状态下,2个偏移角都相等;串联结合的同源二聚体在结合状态下2个偏移角相等。结论:同源二聚体转录因子识别螺旋相对运动存在平面摆动现象。     

人工转录因子研究进展

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结合结构域与效应结构域两部分组成,研究发现这两个结构域可以各自独立发生作用。基于转录因子的这种结构特点,可以人为地选择针对特定序列的DNA结合结构域与具有特定作用的效应结构域构建人工转录因子。目前人工转录因子的DNA结合结构域多为C₂H₂ 型锌指结构,每一个锌指单元由大约30个氨基酸组成,识别DNA双螺旋大沟中相连的3bp序列,并可通过氢键作用与相应的碱基结合;多个锌指可以串联成簇,从而识别并结合较长的DNA序列区域。常见的人工转录因子的效应结构域有激活结构域以及抑制结构域,不同的效应结构域赋予人工转录因子不同的功能。目前人工转录因子已经在基础研究、药物设计以及基因治疗等领域得到了广泛的应用。     

鉴定水稻中一个新的专一结合GCC元件的AP2/EREBP族转录因子

TSH1,是通过搜寻GenBank的EST库而获得的一个来源于水稻的含AP2/EREBP保守结构域的蛋白质.为了详细分析TSH1蛋白与其DNA顺式元件的结合特性,首先采用传统的凝胶阻滞实验和酵母单杂交技术,证实TSH1在体内和体外均专一性地结合于GCC元件,然后利用原子力显微镜技术精确测量了TSH1蛋白与GCC元件在单分子水平的相互作用力.结果表明,GST-TSH1与DRE元件没有特异性的结合,而GST-TSH1与GCC元件结合力的大小为(83.9±2.2)pN,这种特异性的结合可以被加入的游离TSH1蛋白明显降低.GST蛋白和突变GCC元件作为负对照显示出与GCC元件无特异性作用力.以上结果充分证明,TSH1是专一性地与GCC元件相作用的转录因子,而且原子力显微镜对于检测转录因子与DNA相互作用时单碱基的突变十分灵敏.通过比较几种评估转录因子与DNA顺式元件结合特异性的方法,阐述了原子力显微镜技术的特点及优越性.     

细菌GntR家族转录调控因子的研究进展

GntR家族转录调控因子是细菌中分布最为广泛的一类螺旋-转角-螺旋(helix-turn-helix,HTH)转录调控因子,此家族转录调控因子包含两个功能域,分别是N端的DNA结合结构域和C端的效应物结合结构域/寡聚化作用结构域.DNA结合结构域的氨基酸序列是非常保守的,但效应物结合结构域/寡聚化作用结构域的氨基酸序列...     

TBP与TBP相关因子

真核细胞中,一定数量和种类的TBP相关因子与TBP结合成多蛋白复合物--基本起始因子SL1、TFⅡD、TFⅢB,分别指导三类基因的转录.因此,TBP是一种通用转录因子,而TAFs则具有聚合酶和启动子特异性,起辅助转录激活因子的作用.在转录起始过程中,前者在种属间高度保守的C端独特结构可直接识别TATA元件,或通过TAFs与DNA结合;而后者有的作为TBP结合DNA的媒介,有的作为转录起始复合物组装时其他TAFs与TBP相互作用的桥梁,有的则作为转录激活蛋白与TBP联系的纽带,介导转录激活蛋白对基因转录的激活作用.     

转录激活蛋白的分子结构研究进展

在转录激活蛋白分子中,DNA结合区和转录激活区分别位于两个结构域中。DNA结合区有三种基本结构,即螺旋-转折-螺旋结构、锌指结构和亮氨酸拉链结构。DNA结合区的结构特征决定着DNA-蛋白质相互作用的特异性。转录激活区为带负电的亲水性α-螺旋结构,激活作用的强弱取决于激活区的负电性和空间结构。     

植物MYB蛋白与其靶定DNA结合位点之间的相互作用

MYB转录因子是植物转录因子中最大的家族之一,在植物生长发育和对环境胁迫的应激反应中发挥重要作用。MYB蛋白通过识别和结合特定的DNA序列调控靶基因的表达,进而发挥其多样性的作用。近几十年来,MYB蛋白与其靶定DNA结合位点之间的相互作用研究取得了很大的进展。主要综述了植物MYB蛋白与DNA的结合特性及其DNA结合位点的序列特异性,并对检测蛋白质与DNA之间相互作用的新兴技术做了简要阐述。     

FoxA转录因子在肝脏发育中的研究进展

FoxA蛋白是一类DNA结合区具有翼状螺旋结构的转录因子,已发现其三名成员FoxAl、FoxA2和FoxA3在哺乳动物胚胎期的器官形成、成体时期的新陈代谢和内环境稳定中起着重要作用。肝脏发育FoxA亚家族成员起着关键调控作用,在肝向命运决定中扮演“先锋因子”的角色。该文对FoxA转录因子在肝脏发育中的调控作用进行了小结,综述了近年来的最新研究成果。     

哺乳动物转录因子DNA结合谱

基因差异表达是生物发育和对刺激作出应答的分子基础,转录因子在这种基因差异表达中发挥着重要的调控作用。因此,要弄清楚转录因子调控基因差异表达的机理,就必须鉴定出它们全部的靶基因并构建其操纵的转录调控网络。对基因组DNA的序列特异性结合是转录因子调控基因转录的关键环节,因此,要鉴定转录因子的靶基因,就必须从它们与DNA相互作用的分子水平,鉴定它们能够识别并结合的全部DNA序列,即转录因子DNA结合谱。近年来随着DNA微阵列芯片和高通量DNA测序技术的产生和快速发展,出现了建立转录因子体内及体外DNA结合谱的一系列革命性的新技术,对该领域的研究带来重大影响。这些新技术主要包括建立转录因子体内DNA结合谱的染色质免疫沉淀-芯片技术(ChIP-chip)和染色质免疫沉淀-测序技术(ChIP-Seq),以及建立转录因子体外DNA结合谱的双链DNA微阵列芯片技术(dsDNA microarray)、指数富集配体系统进化-系列分析基因表达技术(SELEX-SAGE)、结合-n-测序技术(Bind-n-Seq)、多重大规模并行SELEX技术(MMP-SELEX)、凝胶迁移实验-测序技术(EMSA-Seq)和高通量测序-荧光配体互作图谱分析技术(HiTS-FLIP)。文章将对这些新技术做一综述。     

Watergate技术在DNA上的应用

比较了Watergate和其他几种不同的压水峰方法,结果表明Watergate是效果较好,实验上容易实现的水峰抑制方法.用Watergate-NOESY及Watergate-TOCSY,完成了转录因子E2F所识别的DNA双链片段d(5′-TTTCGCGC)·d(3′-AAAGCGCG)中大部分G,C碱基上可交换质子的归属.     

美达霉素产生菌中立体专一性酮基还原酶基因med-ORF12的表达

美达霉素是链霉菌产生的具有强抗肿瘤活性的芳香聚酮类抗生素,其砒喃环并内酯结构对于其抗癌活性非常重要。位于美达霉素生物合成基因簇中的基因med-ORF12编码立体专一性酮基还原酶,可能参与美达霉素的砒喃环并内酯结构中手性中心(C3S)的形成,但在美达霉素产生菌中的功能和表达还未曾研究。【目的和方法】为了研究med-ORF12在野生菌中的表达情况以及与美达霉素生物合成的关系,本文采用了原核表达、抗体制备、免疫杂交等技术方法对这个基因展开了体内表达研究。【结果】首先利用pET载体建立了med-ORF12的原核表达系统,在优化诱导表达条件的基础上获得了可溶性目的蛋白,制备了相应的多抗血清;然后利用多抗血清对美达霉素产生菌中的基因med-ORF12的表达情况进行了检测,表明在美达霉素产生菌中参与次生代谢的med-ORF12在稳定期大量表达,同时伴随美达霉素的大量积累。【结论】这些结果表明在美达霉素产生菌中,基因med-ORF12参与次生代谢,其表达与美达霉素生物合成有一定相关性。     

Steric Effects on Interaction of Tea Catechins with Lipid Bilayers

Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Tea catechins are classified into cis-type and trans-type from the configuration of the two hydrogens at the 2 and 3 positions on the C-ring. The amount of trans-type catechins incorporated into liposomes was less than that of the respective cis-type catechins. Furthermore, the order of the partition coefficients of catechins in an n-octanol/PBS system is the same as that of the amount incorporated into liposomes. These results indicate that in addition to the number of hydroxyl groups on the B-ring and the presence of the galloyl moiety, the stereochemical structure of the C-ring also governs the hydrophobicity and the affinity for lipid bilayers. Trans-type catechins with the galloyl moiety were located on the surface of the lipid bilayer, as well as cis-type catechins with the galloyl moiety, and perturbed the membrane structure. These different stereochemical structures should influence the affinity for lipid bilayers, the alteraction of membrane structures, and the difference in the order of the biological activities.     

Simple,recurring RNA binding sites for L-arginine

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.     

Stereochemical facets of clinical β-blockers: An overview

The modern β-adrenergic agonists (β-blockers) possess one or more than one chiral center in their structure. Two enantiomers exhibit distinct pharmacodynamic and pharmacokinetic behaviors. Current progress in drug designing has resulted in the ability to understand the role of chirality in modern therapeutics. Furthermore, with a greater understanding of the molecular structure of precise drug targets, development of new drugs is directed towards the pure enantiomers instead of its racemates. The present review deals with a discussion on the stereochemical facets of chiral clinical β-blockers. This review provides details of stereo-selectivity in the pharmacological behavior of some of β-blockers and their metabolites. An effort has been made on highlighting the distinction between the therapeutic behavior of the racemic mixtures and pure enantiomers.     

Isolation of Cycloclavine from the Culture Broth of Aspergillus japonicus SAITO

Changes of protein components (LA, LB, LC, and X) in mouse serum after administration of an antitumor agent, LC 9018 (lyophilized preparation of Lactobacillus casei, YIT 9018), were investigated. The intraperitoneal injection of LC 9018 caused an increase in these proteins one or two days after administration. The LC component was purified from mouse serum and was identified as haptoglobin. In addition, LA was identified as an intermediate of a haptoglobin-hemoglobin complex [Hp-Hb(α₁β₁)]and X as a haptoglobin-hemoglobin complex [Hp-Hb(α₂β₂)] because addition of increasing amount of hemoglobin to purified haptoglobin formed X via LA and all of these components were immunoprecipitated with anti-haptoglobin IgG.     

Geometry of guanidinium groups in arginines

The restraints in common usage today have been obtained based on small molecule X‐ray crystal structures available 25 years ago and recent reports have shown that the values of bond lengths and valence angles can be, in fact, significantly different from those stored in libraries, for example for the peptide bond or the histidine ring geometry. We showed that almost 50% of outliers found in protein validation reports released in the Protein Data Bank on 23 March 2016 come from geometry of guanidine groups in arginines. Therefore, structures of small molecules and atomic resolution protein crystal structures have been used to derive new target values for the geometry of this group. The most significant difference was found for NE‐CZ‐NH1 and NE‐CZ‐NH2 angles, showing that the guanidinium group is not symmetric. The NE‐CZ‐NH1 angle is larger, 121.5(10)?, than NE‐CZ‐NH2, 119.2(10)?, due to the repulsive interaction between NH1 and CD1 atom.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

Absolute configuration of (-)-2,6-dimethyl-10-(p-tolyl)-2,6(E)-undecadiene from Cistus monspeliensis

(-)-2,6-Dimethyl-10-(p-tolyl)-2,6(E)-undecadiene (1) is a major constituent in the essential oil of Cistus monspeliensis, an aromatic shrub common in Mediterranian countries. 1 was isolated by column chromatography, subjected to ozonolyses, and the absolute configuration was determined by enantioselective gas chromatographic correlation with the ozonolysis product of the sesquiterpene hydrocarbon ar-curcumene with known absolute configuration.     

Side-chain rotamers in protein α-α hairpins and a mechanism of their selection

Several regularities were observed for the distribution of side-chain rotamers in α-α hairpins of globular proteins. In left-turned α-α hairpins, most side chains adopt t rotamers in d-positions and g^? rotamers in g-positions. In right-turned α-α hairpins, most side-chains adopt g^? rotamers in a-positions and t rotamers in e-positions. Analysis of these regularities suggested that selection of the side-chain conformation in α-α hairpins depends on two main factors, the mode of α-helix packing and the positions of side chains in α-helices. The regularities were explained by the squeezing mechanism: interhelical interactions bring the α-helices close together so that the side chains are squeezed out of the helix-helix interface and adopt unique conformations.     

Role of the structural context in selection of hydrophobic side-chain rotamers in a- and d-positions of α-helices

It was demonstrated for the first time that the distribution of side-chain rotamers in the a-and d-positions of α-helices of coiled-coil (cc) proteins follows a certain trend, rather then being random. For instance, most side chains adopt t rotamers in the a-positions and g^? rotamers in the d-positions of helical dimers. Vice versa, most side chains adopt g^? rotamers in the a-positions and t rotamers in the d-positions of tetramers. It was concluded that selection of the side-chain rotamers depends on the packing of α-helices and, consequently, depends on the structural context.