锌指蛋白的核酸识别特异性研究进展

锌指蛋白是最大的DNA结合蛋白,它能和DNA进行特异性识别,是研究蛋白－DNA相互作用的理想对象。改变锌指元件上的几个保守的氨基酸位点可设计筛选出序列特异的全新锌指蛋白,计算机在锌指蛋白设计方面的应用,使得全新的锌指蛋白识别特异性明显增强。这在基因治疗等方面,具有广阔的应用前景。     

锌指蛋白的核酸识别特异性研究进展

锌指蛋白是最大的DNA结合蛋白,它能和DNA进行特异性识别,是研究蛋白—DNA相互作用的理想对象。改变锌指元件上的几个保守的氨基酸位点可设计筛选出序列特异的全新锌指蛋白,计算机在锌指蛋白设计方面的应用,使得全新的锌指蛋白识别特异性明显增强。这在基因治疗等方面,具有广阔的应用前景。     

结合于GATA元件的Ⅳ型锌指蛋白

至今已发现了四种锌指蛋白（Ｚｆｐ）。Ⅰ型为Ｃｙｓ－Ｘ２－４－Ｃｙｓ－Ｘ３－Ｐｈｅ－Ｘ５－Ｌｅｕ－Ｘ２－Ｈｉｓ－Ｘ３－Ｈｉｓ,简写Ｃ２Ｈ２。ＴＦⅢＡ为Ｃ２Ｈ２×９,即９个锌指的重复单位。Ｓｐ１为Ｃ２Ｈ２×３。目前已发现有１０００种以上具有Ⅰ型锌指同源保...     

“锌指”结构:蛋白质和DNA相互作用的一种模式

锌指结构是DNA结合蛋白的基本模型之一,它广泛存在于真核细胞与基因调控有关的蛋白质中。文章综述了锌指蛋白的发现、存在、结构模型、与DNA结合特点和生物功能,以及近期的研究重点。     

锌指蛋白的设计及其应用

人工设计的锌指蛋白一般包括两个结构域：DNA结合结构域和效应结构域。DNA结合结构域主要采用对DNA序列特异性识别结合的C2H2型锌指结构域,而功能结构域常常采用某些转录激活结构域、转录抑制结构域或某些酶的活性结构域。这样进行设计的锌指蛋白就可以在特定的核酸序列上行使相应的功能,这对于目的基因的表达调控及蛋白质与核酸的相互作用研究提供了新的思路。     

C2H2型锌指蛋白的研究进展

锌指基因家族是人体中最大的基因家族,它参与细胞分化、胚胎发育,并与许多疾病的发生相关．根据半胱氨酸(c)和组氨酸(H)的数目和位置可将锌指蛋白分为c2H2、c2Hc2、c2c2 CHCC2C2、C2C2C2C2等亚类．c2H2型锌指是最普遍的类型,它们作为重要的转录调控因子参与许多的生理过程．c2H2型锌指蛋白包含的锌指数目从1个到30多个不等．依据锌指的数量以及在蛋白中的分布情况,大多数c2H2型锌指蛋白属于下列3类之一：1)含3个c：H：锌指的蛋白(tC2H2);2)含多个锌指的c2H2型锌指结构蛋白(mac2H2);3)锌指成对间隔排列的c2H2型锌指蛋白(spC2H2)、一些c2H2型锌指蛋白能识别并结合特异性RNA或DNA片段．另一些则只能与RNA结合．通常锌指蛋白含锌指数目越多。它选择结合的能力就越强．     

锌指蛋白结构及功能研究进展

锌指蛋白是一类具有手指状结构域的转录因子,对基因调控起重要的作用。根据其保守结构域的不同,可将锌指蛋白主要分为C2H2型、C4型和C6型。锌指通过与靶分子DNA、RNA、DNA-RNA的序列特异性结合,以及与自身或其他锌指蛋白的结合,在转录和翻译水平上调控基因的表达。我们简要综述了近年来锌指蛋白结构、分类及其与核酸及蛋白质相互作用等方面的研究进展。     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

The extended left-handed helix: a simple nucleic acid-binding motif

The poly-proline type II extended left-handed helical structure is well represented in proteins. In an effort to determine the helix's role in nucleic acid recognition and binding, a survey of 258 nucleic acid-binding protein structures from the Protein Data Bank was conducted. Results indicate that left-handed helices are commonly found at the nucleic acid interfacial regions. Three examples are used to illustrate the utility of this structural element as a recognition motif. The third K homology domain of NOVA-2, the Epstein-Barr nuclear antigen-1, and the Drosophila paired protein homeodomain all contain left-handed helices involved in nucleic acid interactions. In each structure, these helices were previously unidentified as left-handed helices by secondary structure algorithms but, rather, were identified as either having small amounts of hydrogen bond patterns to the rest of the protein or as being "unstructured." Proposed mechanisms for nucleic acid interactions by the extended left-handed helix include both nonspecific and specific recognition. The observed interactions indicate that this secondary structure utilizes an increase in protein backbone exposure for nucleic acid recognition. Both main-chain and side-chain atoms are involved in specific and nonspecific hydrogen bonding to nucleobases or sugar-phosphates, respectively. Our results emphasize the need to classify the left-handed helix as a viable nucleic acid recognition and binding motif, similar to previously identified motifs such as the helix-turn-helix, zinc fingers, leucine zippers, and others.     

Fractionation with ethanol of nucleic acids from viroid-infected plants

A combination of high salt and low ethanol concentration allowed the fractionation of nucleic acids extracted from viroid-infected leaves. By adding 0.4-0.5 vol of ethanol to 1 vol of a solution in 2 M LiCl of nucleic acids (containing mainly DNA, 4S, 5S, 7S, and viroid RNAs), 85% of the DNA and 75% of the 4S RNA remained in solution, from where they could be recovered by increasing the ethanol concentration, whereas almost all 5S, 7S, and viroid RNAs precipitated. When this process was repeated three times a 95% elimination of the initial DNA and 4S RNA was achieved. The method can be of special interest in viroid purification considering that DNA and 4S RNA are the most abundant contaminants in the starting solution of nucleic acids. It is suggested that the highly ordered secondary structure of viroid RNA may be responsible for its particular behavior in the ethanol fractionation of nucleic acids.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

蛋白质/核酸相互作用研究方法进展

蛋白质和核酸是构成生命体最为重要的两类生物大分子,蛋白质与核酸的相互作用是分子生物学研究的中心问题之一,它是许多生命活动的重要组成部分。研究蛋白质/核酸相互作用近期采用的新技术有:核酸适体技术、生物信息学方法、蛋白质芯片技术以及纳米技术等。本文就这些新的研究方法进行综述。     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.     

qNABpredict: Quick,accurate, and taxonomy-aware sequence-based prediction of content of nucleic acid binding amino acids

Protein sequence-based predictors of nucleic acid (NA)-binding include methods that predict NA-binding proteins and NA-binding residues. The residue-level tools produce more details but suffer high computational cost since they must predict every amino acid in the input sequence and rely on multiple sequence alignments. We propose an alternative approach that predicts content (fraction) of the NA-binding residues, offering more information than the protein-level prediction and much shorter runtime than the residue-level tools. Our first-of-its-kind content predictor, qNABpredict, relies on a small, rationally designed and fast-to-compute feature set that represents relevant characteristics extracted from the input sequence and a well-parametrized support vector regression model. We provide two versions of qNABpredict, a taxonomy-agnostic model that can be used for proteins of unknown taxonomic origin and more accurate taxonomy-aware models that are tailored to specific taxonomic kingdoms: archaea, bacteria, eukaryota, and viruses. Empirical tests on a low-similarity test dataset show that qNABpredict is 100 times faster and generates statistically more accurate content predictions when compared to the content extracted from results produced by the residue-level predictors. We also show that qNABpredict's content predictions can be used to improve results generated by the residue-level predictors. We release qNABpredict as a convenient webserver and source code at http://biomine.cs.vcu.edu/servers/qNABpredict/ . This new tool should be particularly useful to predict details of protein–NA interactions for large protein families and proteomes.