三十年磨一剑———2006年诺贝尔化学奖得主Roger Kornberg关于真核基因转录机制的研究

2006年的诺贝尔化学奖授予了美国斯坦福大学的科恩伯格（Roger D．Komberg）教授,以表彰他在真核基因转录的分子机制研究方面做出的卓越贡献。在细胞中,DNA的复制、RNA的转录和蛋白质的翻译是生命活动的核心过程。因此,研究遗传信息如何从DNA到RNA再到蛋白质的传递过程一直是分子生物学研究的核心课题。科恩伯格的工作则在分子水平上向我们展示了RNA聚合酶Ⅱ（RNA polymerase Ⅱ）及各种蛋白因子在DNA模板上合成作为蛋白质合成模板的信使RNA（mRNA）的过程,为在转录水平阐明真核基因的表达调控奠定了分子结构基础。     

DNA复制、转录及蛋白质生物合成的微机程序设计

核酸及蛋白质是现代生物研究的重点,是生物化学、分子遗传学、分子生物学等的主要内容。为了配合教学,开展微机应用,设计了DNA复制、转录及蛋白质生物合成的电子计算机程序。当输入一定的DNA(模板DNA,方向为3′→5′)序列时,通过此程序运行,可分别准确地打印出与模板DNA对应的DNA互补链(复制),特定核苷酸排列的mRNA链(转录)及其由特定氨基酸排列的多肽链(翻译)。     

无细胞蛋白质合成系统的研究进展及应用前景

无细胞蛋白质合成系统是现代迅速发展的蛋白质表达系统,以细胞抽提物中酶和蛋白质因子等作为基本反应体系,添加外源模板、底物以及能源物质等维持体系运作,最终在体外合成目标蛋白质。无细胞蛋白质合成系统突破了细胞的生理限制,能够灵活地进行蛋白质合成,极大地提高了蛋白质的产量,不仅可以作为研究转录和翻译的研究工具,而且可以实现蛋白质的高通量表达,在诸多领域带来了突破性的进展。现主要针对无细胞蛋白质合成系统的能量供给、蛋白质的稳定性和折叠修饰以及应用进行综述,以期推动对该技术的理解和应用。     

关于脑缺血的分子生物学研究

脑缺血的主要直接后果是脑缺氧。脑缺血或脑缺氧除引起一系列神经化学变化与蛋白质的合成降低外,还引起热休克蛋白（ＨＳＰ）和ｃ－ｆｏｓ蛋白等特殊蛋白质的特殊变化。轻度缺血可引起ＨＳＰ７０基因转录与翻译;中度缺血只引起其转录而不翻译;重度缺血时转录与翻译均终止。轻、中度脑缺血引起ｃ－ｆｏｓｍＲＮＡ剧烈而短暂的表达,ｃ－ｆｏｓ、ＪｕｎＢ、ｃ－ｊｕｎ等基因转录增加;严重缺血可能因神经元死伤,导致ｃ－ｆｏｓ蛋白水平降低,但局部胶质细胞ｃ－ｆｏｓ诱导增强。     

转录 反转录

有机体的遗传信息一般都编码在由缠绕成双螺旋的两条长链所组成的脱氧核糖核酸(DNA)分子上,由四个码编成,这四个码是不同的化学单位,叫做碱基。在正常细胞中要合成某种蛋白质,遗传信息是以DNA为模板,根据碱基互补的原则合成与它对应的单链分子核糖核酸(RNA),然后再从RNA链译成特定的蛋白质分子。即由DNA→RNA→蛋白质。由DNA→RNA称为“转录”,由RNA→蛋白质称为“翻译”。反转录是遗传信息以RNA为模板合成DNA,即同上述信息的转移从DNA→RNA这一经典过程相反,因此称“反转录”。例如,在RNA肉瘤病毒进入机体后,通过依赖于病毒RNA的DNA多聚酶,以病毒RNA为模板合成DNA,然后再以DNA     

真核基因启动子非依赖的功能转录延伸复合物的体外组装

真核生物RNA聚合酶Ⅱ的持续合成能力对基因转录过程中每一个阶段,包括启动子脱离、转录暂停、转录终止以及转录偶联DNA损伤修复过程的调节至关重要.在RNA聚合酶Ⅱ介导的转录延伸过程中,其和模板DNA及转录产物RNA紧密结合,形成一个非常稳定的延伸三维复合物(elongationcomplex,EC).此特征性“泡”状结构的形成是RNA聚合酶Ⅱ持续合成能力所必需的.在不依赖启动子及众多转录起始因子的条件下,利用人工合成的RNA与DNA寡核苷酸,在体外组装形成具有功能转录活性的延伸复合物.结果表明,长度为9个核苷酸的RNA与模板DNA形成的杂合分子对转录延伸复合物的形成是必需的,而非转录模板DNA链的加入导致最终活性转录“泡”状复合物的形成,并可转录形成与模板相关的转录产物,进一步通过在模板DNA的特定位置引入一个乙酰氧乙酰氨基芴修饰基团,可特异性地阻断转录延伸过程,从而显示该系统在研究真核基因转录及转录偶联DNA损伤修复机制中的潜在应用价值.     

体外翻译系统在分子生物学研究中的应用

体外翻译系统又称无细胞蛋白质合成系统,是分子生物学中一种常规的表达系统,该系统可用于蛋白质快速分析鉴定,基因转录和翻译的调控机理的研究以及分子间的相互作用的研究,如蛋白质和蛋白质的相互作用,蛋白质与DNA的相互作用,蛋白质与RNA的相互作用等。     

反义及正义表达癌基因c-Ha-ras重组质粒的构建

c-Ha-ras癌基因通过其产物p21蛋白的点突变或过量表达使细胞恶变。国外一些实验室的初步研究表明,导入一段与c-Ha-ras基因转录出的mRNA(sense RNA,正义RNA)顺序互补的RNA(anti-sense RNA,反义RNA),有可能通过阻断DNA复制,RNA转录或蛋白质翻译等过程,减少p21蛋白的合成,从而使转化细胞逆转。为了进一步系统研究反义c-Ha-ras RNA在转化细胞逆转中的     

细菌合成蛋白质量控制系统

2021年2月5日Nature Chemical Biology报道,韩国首尔国立大学工程学院研究团队开发了一个合成蛋白质质量控制系统(protein quality control,ProQC),可以增强细菌的蛋白质全长翻译能力。重组蛋白质已经广泛应用于各种工业领域。蛋白质需要保持全长和适当的三维结构才能发挥功能。但是,由于细菌中的转录和翻译步骤同时发生在同一个地方,截短的基因可以作为核糖体翻译的模板,从而产生不完整的多肽。     

逆转录现象的发现及其意义

现代遗传学已经证明,I）NA是生物遗传的主要物质基础。生物体的遗传特征是以遗传密码的形式编码在＊＊A分子上,表现为特定的核音酸排列顺序,并且通过DNA的复制,把遗传信息由亲代传递给予代。在后代的个体发育中,遗传信息由DNA转录给RNA,然后通过mRNA翻译合成特异的蛋白质以执行各种生命功能,从而使后代表现出与亲代相似的遗传性状。这就是本世纪SO年代末所确定的蛋白质合成的“中心法则”。“中心法则”确定后,人们发现并不是所有RNA都是在DNA模板上复制的。许多病毒并没有DNA,只有单链的RNA作为遗传物质。当这些病毒…     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Regional distribution of S-100a0 (αα), S-100a (αβ) and S-100b (ββ) in the bovine central nervous tissue determined with a sensitive enzyme immunoassay system

A sensitive sandwich-type enzyme immunoassay system for separate measurement of 3 forms of bovine S-100 protein, S-100a₀ (αα), S-100a (αβ) and S-100b (ββ), was developed by the use of purified antibodies to the α or the β subunit of bovine S-100 protein. The assay system consisted of polystyrene balls with immobilized antibody (anti-α for S-100a₀ and S-100a assays, and anti-β for S-100b assay) F(ab′)₂ fragments and antibody (anti-α for S-100a, assay, and anti-β for S-100a and S-100b assays) Fab′ fragments labeled with β-d-galactosidase from Escherichia coli. The minimum measurable sensitivity of each assay was less than 10 pg/assay tube. The assay system for S-100a cross-reacted little with S-100a₀ and S-100b. The assay systems for S-100a₀ and S-100b cross-reacted (10 and 17%, respectively) with S-100a which contains α and β subunits in the molecule. However, levels of S-100a₀, S-100a and S-100b in the soluble extract of bovine brain could be determined by correcting the cross-reacted S-100a to the assays of S-100a₀ and S-100b. Various regions of bovine central nervous tissue were found to contain 0.3–1 μg of S-100a₀, 4–14 μg of S-100a, and 8–30 μg of S-100b per mg soluble protein. The percent concentrations of three forms of S-100 protein in the cerebral cortex were about 3, 38, and 59, for S-100a₀, S-100a, and S-100b, respectively, and those in the cerebellar cortex were 2, 21 and 77, respectively. Purified S-100a and S-100b preparations from human and rat brains were also reactive with the respective assay system for bovine S-100 protein, suggesting that the present assay system is applicable to the assay of three forms of S-100 protein in human and rat tissues.     

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Defective protein synthesis of S-30 fraction of reticulocytes of anemic b/b rats.

The preincubated S-30 fraction from reticulocytes of anemic b/b rats is not able to promote the synthesis of proteins in the presence of hemoglobin RNA provided either from the reticulocytes of phenylhydrazine-treated rats or from the reticulocytes of anemic b/b rats. It was observed that poly(U)-directed polyphenylalanine synthesis is negligible in the same system, suggesting the existence of some alteration in regulatory machinery for protein synthesis in the reticulocytes of the anemic b/b rats.