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1.
硒蛋白的生物合成与调控   总被引:4,自引:0,他引:4  
硒蛋白是硒以硒半胱氨酸(Sec)形式参入形成的蛋白质。Sec作为参入蛋白质的第21种氨基酸,由硒蛋白mRNA上的UGA编码。在原核生物中,Sec参入硒蛋白的相关因子及其参入机制已基本阐明,Sec在SELA、SELB、SELC、SELD及Sec插入序列(SECIS)等的共同作用下参入到蛋白质中。在真核生物中,Sec参入硒蛋白的可能途径是:Ser-tRNA‘^[Ser]Sec。通过磷酸丝氨酰-tRNA^[Ser]Sec。最终转变为Sec-tRNA^[Ser]Sec,并在延伸因子及相关蛋白质因子的作用下参入到硒蛋白中。硒蛋白的合成在翻译前水平、mRNA水平、供硒水平等都受到相应的调控。  相似文献   

2.
生物合成硒蛋白机制的研究进展   总被引:8,自引:0,他引:8  
作为第 2 1种氨基酸 ,硒代半胱氨酸在翻译阶段由核糖体介导 ,在mRNA编码区的UGA密码子处参入多肽链。研究表明硒代半胱氨酸的参入需要一个顺式作用元件SECIS和 4个基因产物 :SelA、SelB、SelC、SelD。原核生物和真核生物的SECIS在mRNA中的位置和结构特征差异显著。在利用Escherichiacoli硒代半胱氨酸的参入机制合成硒蛋白方面 ,研究人员进行了有益的探索。  相似文献   

3.
在原核生物中,硒蛋白合成需要tRNA~(Sec) (SelC)与硒代半胱氨酸合成(Sec synthase, SelA)、硒代半胱氨酸特异性延伸因子(Sec-specificelongationfactor,SelB)之间相互作用。【目的】基于大肠杆菌掺硒机器,寻找tRNA~(Sec)骨架上关键核苷酸位点,为解决硒蛋白目前面临的掺硒效率较低、产量低的问题提供新思路。【方法】以大鼠细胞质型硫氧还蛋白还原酶(thioredoxinreductase1,TrxR1)为掺硒模式蛋白为定点突变tRNA~(Sec),转化至BL21 (DE3) gor-获得阳性重组菌株(携带pET-TRSter/pSUABC’),用于表达大鼠硒蛋白TrxR1,然后使用2¢,5¢ADP-Sepharose亲和层析和凝胶过滤两步法分离纯化TrxR1,最后利用经典硒依赖型DTNB还原反应测定TrxR1的酶活,分析关键核苷酸位点,评价掺硒效率。【结果】在存在SECIS元件的前提下,当SelA、SelB、tRNA~(Sec)共表达时,与野生型相比,携带突变型tRNA~(Sec)所共表达的TrxR1酶活力呈现不同程度的降低,其中E.colitRNA~(Sec)的G18、G19这两个位点的所有的TrxR1酶活远低于野生型(10%);然而,a26和b7的酶活相对较高。【结论】E. coli tRNA~(Sec)骨架上G18和G19位点对于维持tRNA稳定性和灵活性发挥了关键作用,位点突变引起tRNA结构变化会影响tRNA~(Sec)与掺硒元件的互作,因此有望通过改造tRNA核苷酸位点来提高硒蛋白的掺硒效率。  相似文献   

4.
硒蛋白P的研究进展   总被引:4,自引:0,他引:4  
硒蛋白P(SeP)是从大鼠和人血浆中分离,纯化得到的一种糖蛋白,每个硒蛋白P多肽含有10个硒代半胱氨酸,硒蛋白P中的硒含量占大鼠和人血浆中硒含量的50%以上,在其mRNA开放阅读框架中克隆的cDNA的序列含有10个UGA密码子。硒代半胱氨酸在一个UGA密码子处嵌入蛋白的一级结构,尽管对硒蛋白P功能还没有彻底了解,它的一种非常可能的作用是作为一种胞外抗氧化剂,大鼠血浆中的硒蛋白P在体内实验中对Diquat诱导的脂质过氧化和肝损坏具有保护作用,人血浆中的硒蛋白P在体外实验中显示减少内作为一存活促进因子。  相似文献   

5.
硒蛋白是一类含有硒代半胱氨酸(Selenocysteine,Sec)的蛋白质,具有抗氧化和免疫调节等多种生物学功能。尽管目前已知硒对鱼类生长发育、抗氧化和免疫等方面有着重要的作用,但是有关鱼类硒蛋白的研究还不全面,特别是对鱼类硒蛋白W(Selenoprotein W,Sel W)的结构和功能还知之甚少。为了深入研究鱼类硒蛋白的结构特征和生理作用,本文利用RT-PCR和RACE技术从肝胰脏中克隆获得了鲤鱼Cyprinus carpio Sel W的c DNA全长序列(cc Sel W)。cc Sel W c DNA全长704 bp,含有261 bp的开放阅读框,编码86个氨基酸。预测的cc Sel W分子量为9.5 k D,在其N-端含有特征序列C-X-X-U基序,且第13位氨基酸为13Sec。在cc Sel W c DNA 3'-UTR区域含有一个89 bp的Sec插入序列(Sec-insertion sequence,SECIS),形成经典的AUGA-AA-GA型SECIS。由于在其SECIS二级结构的顶环上有一个额外的小茎-环结构,所以鲤鱼SECIS属于Ⅱ型SECIS结构。cc Sel W与斑马鱼Danio rerio和热带爪蟾Xenopus tropicalis的同源性最高,均为84.7%,与其他物种的同源性在54%~60%之间。系统发育树结果显示,cc Sel W和斑马鱼、两栖类位于同一分支上。RT-PCR结果显示,cc Sel W mRNA在所检测的11个组织中均有表达,其中以精巢中的表达量为最高,下丘脑和肠中表达量次之,其余组织中表达量大致相当,低于精巢、下丘脑和肠。这些结果丰富了鱼类Sel W的研究内容,为进一步研究其结构和功能奠定了基础。  相似文献   

6.
硒蛋白P的研究进展   总被引:2,自引:0,他引:2  
硒蛋白P最初在血浆中发现,占血浆总硒的60%,在其多肽链中有10个硒半胱氨酸。由10个读码框内的UGA编码,而UGA一般是作为终止密码子起作用,故硒蛋白P的生物合成需要多个特异的因子。如特异的tR-NA,延伸因子和mRNA上的特异的二级结构等。硒蛋白P的功能尚不清,初步的研究结果提示可有转运硒,抗氧化,结构重金属和神经营养等作用。  相似文献   

7.
硒蛋白P的研究进展   总被引:1,自引:0,他引:1  
硒蛋白P(SeP)是从大鼠和人血浆中分离、纯化得到的一种糖蛋白 ,每个硒蛋白P多肽含有10个硒代半胱氨酸。硒蛋白P中的硒含量占大鼠和人血浆中硒含量的 5 0 %以上。在其mRNA开放阅读框架中克隆的cDNA的序列含有 10个UGA密码子。硒代半胱氨酸在一个UGA密码子处嵌入蛋白的一级结构 ,尽管对硒蛋白P功能还没有彻底了解 ,它的一种非常可能的作用是作为一种胞外抗氧化剂。大鼠血浆中的硒蛋白P在体内实验中对Diquat诱导的脂质过氧化和肝损坏具有保护作用 ,人血浆中的硒蛋白P在体外实验中显示减少内毒素过氧化硝酸盐和磷脂氢过氧化物的活性。牛血浆中的硒蛋白P在神经细胞的培养中作为一存活促进因子。  相似文献   

8.
疟蚊基因组中新硒蛋白的计算机识别   总被引:5,自引:0,他引:5  
硒蛋白的生物合成取决于硒代半胱氨酸插入蛋白质的过程. TGA码既是终止码、又可翻译成硒代半胱氨酸, 这使普通基因注释软件无法正确预测硒蛋白, 导致现有数据库中许多物种的硒蛋白被错误注释或丢失. 本研究基于已公布的疟蚊基因组预测信息、采用PERL语言编程, 对疟蚊基因组中的硒蛋白进行了计算机检索与分析. 结果表明: 以TGA码终止的基因有11365条, 其中具有SECIS结构的基因有918条, 同时具有半胱氨酸同源类似物的基因58条. 再经Sec侧翼序列比对, 最终检索到具有硒蛋白全部特点的基因7条. 从硒蛋白的基本生物功能推测, 冈比亚疟蚊基因组中存在的新硒蛋白可能与疟蚊的氧化耐受特性及其相关蛋白的调控相关联. 因此, 研究疟蚊硒蛋白将为干涉疟蚊带菌能力、从蚊媒传播途径防止疟疾提供理论基础.  相似文献   

9.
硒蛋白     
硒(Se)已被确认为是一种生物微量元素,它能共价结合到生物大分子、尤其是蛋白质中。硒蛋白是某些细菌、鸟类、哺乳动物(可能也包含植物)的酶系统的基本成份。一、细菌硒蛋白最早被鉴定的细菌硒蛋白是依赖硒的甲酸脱氢酶,该酶催化无氧条件下HCOOH?H_2+CO_2。Pinsent(954)指出,E·Coli甲酸脱氢酶的表达需要硒。Lester和Demoss(1971)则  相似文献   

10.
硒酶及硒化合物生理功能研究的新进展   总被引:17,自引:0,他引:17  
硒是动物必需的微量元素,硒半胱氨酸是硒蛋白的组份并构成硒酶的活性中心,硒蛋白特别是硒酶是硒的主要功能形式,研究发现,硒半胱氨酸是参入到蛋白质分子中的第21种氨基酸,硒是唯一受基因调控的微量元素,最新研究表明,硒及其化合物还具有阻断某些炎症介质的生理活性,抑制蛋白激酶C,激活促分裂原活化蛋白激酶和S6核糖体蛋白激酶,免疫调节及与其它元素和维生素相互作用等多种生理功能,提示硒在人类健康中的作用及其机制比我们过去所预计的更为复杂。  相似文献   

11.
Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.  相似文献   

12.
Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.  相似文献   

13.
In mammals, most of the selenium contained in the body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is typically recognized as a translation stop signal, it is intriguing how a cell recognizes and distinguishes a UGA Sec codon from a UGA stop codon. For eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated the Sec insertion sequence (SECIS) in the 3'-untranslated (3'-UTR) region is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human glutathione peroxidase (GPx) mRNA. We found a protein which strongly bound to the RNA fragment upstream of the UGA Sec codon. However, this protein did not bind to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. Comparison of the RNA fragment with the SECIS fragment identified the conserved regions, which appeared in the region upstream of the in-frame UGA Sec codon of Se-protein mRNAs. Thus, this study proposes a novel model to understand the mechanisms of Sec incorporation at the UGA Sec codon, especially the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF of the peptide releasing factor.  相似文献   

14.
Squires JE  Berry MJ 《IUBMB life》2008,60(4):232-235
Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently.  相似文献   

15.
A selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.  相似文献   

16.
17.
Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.  相似文献   

18.
For eukaryotic selenoprotein mRNAs, it has been proposed that the SECIS element in the 3'-UTR is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still being considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human GPx mRNA. We found a protein, prepared from bovine brain microsomes, which strongly bound to the RNA fragment upstream of the UGA Sec codon but not to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. We also obtained the similar results with the RNA fragments of type I iodothyronine 5'-deiodinase (5'DI) mRNAs. Comparison of such RNA fragments with SECIS fragments revealed similarities in the region upstream of the in-frame UGA Sec codon of several Se-protein mRNAs. The study thus favors a novel model of Sec incorporation at the UGA Sec codon that involves the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF.  相似文献   

19.
In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.  相似文献   

20.
Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.  相似文献   

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