游仆虫重组核糖体蛋白L11与肽链释放因子的体外相互作用分析

核糖体蛋白L11(ribosome protein L11)是一种高度保守的蛋白质．为研究真核生物的核糖体蛋白L11的功能,从八肋游仆虫（Euplotes octocarinatus）大核基因组中克隆到核糖体蛋白L11基因,构建了重组表达质粒pGEX-6p1-L11,通过谷胱甘肽-Sepharose 4B亲和层析,纯化了重组融合蛋白GST-L11．Pull down 分析显示,八肋游仆虫的核糖体蛋白L11与第一类肽链释放因子eRF1a可以在体外相互作用．这一结果提示,与原核生物一样,低等真核生物的核糖体蛋白L11在肽链终止过程中可能起一定的作用．     

八肋游仆虫第二类释放因子基因的克隆与序列分析

分离八肋游仆虫 (Euplotesoctocarinatus)大核eRF3基因 ,为进一步研究第二类释放因子结构与功能 ,探讨低等真核生物新生肽链释放机理提供实验素材 .以八肋游仆虫基因组DNA为材料 ,根据已知的第二类释放因子eRF3保守氨基酸序列设计引物 ,扩增克隆了该游仆虫的第二类释放因子基因片段 ,并对其核苷酸序列进行了分析 .根据测得的序列设计特异性引物 ,并利用游仆虫的端粒序列 (C4 A4 C4 A4 C4 A4 C4 )为引物 ,扩增得到该基因的全序列 .序列分析表明 ,该基因位于 2 782bp长的大核染色体上 ,编码区由 2 4 0 0bp组成 ,编码 80 0个氨基酸 ,不含内含子     

游仆虫第一类肽链释放因子在大肠杆菌中的表达、纯化和鉴定

为对肽链释放因子结构与功能进行研究 ,进而探讨纤毛虫这类生物中遗传密码表达特殊性的机理 ,利用PCR技术和基因重组技术构建了游仆虫第 1类肽链释放因子eRF1a及C端带 6个组氨酸的eRF1a(His) 6的两个重组表达质粒pBV2 2 1 eRF1a和pBV2 2 1 eRF1a(His) 6.在大肠杆菌DH5α中 ,通过 4 2℃高温诱导 3h ,eRF1a和eRF1a(His) 6获得了可溶性表达 .eRF1a(His) 6的表达水平达到可溶性细菌总蛋白约 8% ,经Ni NTA亲和层析和HitrapQ离子交换层析 ,得到纯度较好的eRF1a(His) 6.Western印迹鉴定为阳性     

游仆虫EoRab2a蛋白的表达、纯化及定位分析

Rab GTPase家族蛋白是真核细胞内膜系统转运途径中重要的调控因子,不同的Rab家族成员在细胞具有功能多样性。为了解Rab2的功能,八肋游仆虫EoRab2a基因连接入原核表达质粒pGEX-6P-1中,获得重组表达质粒pGEX-6P-1-EoRab2a。质粒pGEX-6P-1-EoRab2a转化大肠杆菌BL21(DE3),经IPTG诱导,大肠杆菌BL21(DE3)/pGEX-6P-1-EoRab2a高效表达了可溶性GST-EoRab2a蛋白。融合蛋白GST-EoRab2a经亲和层析获得电泳纯蛋白。纯化后的GST-EoRab2a免疫BALB/c小鼠制备多克隆抗体。ELISA和Western blotting检测显示制备的抗体效价1∶25600,特异性良好。免疫荧光定位表明EoRab2a在游仆虫细胞质中点状分布,推测参与内质网与高尔基体间膜泡转运。       

八肋游仆虫第二类肽链释放因子核定位信号分析

蛋白质合成终止过程中肽链释放因子负责终止密码子的识别.真核生物第二类肽链释放因子（eRF3）是一类GTP酶,协助第一类肽链释放因子（eRF1）识别终止密码子和水解肽酰 tRNA酯键.之前的研究表明,两类肽链释放因子在细胞核中发挥功能,参与蛋白质合成和纺锤体的组装.本研究根据软件预测结果,构建了一系列八肋游仆虫eRF3的截短型突变体,分析在其N端是否存在引导eRF3的核定位信号.结果表明,在eRF3的N端有两个区域（NLS1:23-36 aa 和 NLS2: 236-272 aa）可以引导eRF3进入细胞核中,而且这两个区域具有典型的核定位信号的氨基酸序列特征. eRF3的核定位与其作为一种穿梭蛋白的功能相一致,即参与细胞有丝分裂纺锤体的形成和无义介导的mRNA降解途径.     

EoRab43参与游仆虫细胞内大核周围的物质运输

Rab家族蛋白是真核细胞内膜泡运输途径中重要的调节因子。EoRab43是八肋游仆虫中一种编码非典型Rab蛋白的基因。本研究依据已获得的EoRab43基因序列设计引物．从八肋游仆虫大核DNA中扩增了EoRab43基因的3’端153bp片段,即EoRab43 153bp（对应于EoRab43蛋白的C末端50个氨基酸,EoRab43C）,构建重组表达质粒pGEX—EoRab43,53bp转化大肠杆菌BL21（DE3）进行表达．纯化后的融合蛋白GST—EoRab43C免疫BALB／c小鼠制备多克隆抗体。经检测,制备的抗体具有较高的效价及良好的特异性。利用制备的抗体对EoRab43在游仆虫细胞内进行免疫荧光定位．结果显示该蛋白主要定位于该生物细胞内大核染色体的周围。     

肽链释放因子和核糖体蛋白L11在八肋游仆虫细胞中的定位

原生动物纤毛虫是一类单细胞真核生物,其蛋白质合成终止过程中密码子使用的特殊性使其成为研究蛋白质合成终止机制的一个经典模型。为了能够有效地分析生物大分子在该细胞中的功能作用位点,本研究根据该生物染色体结构的特征,构建了含有红色荧光蛋白基因的大核人工染色体EoMAC_R,并与之前构建的含绿色荧光蛋白基因的大核染色体EoMAC_G一起,对蛋白质合成终止有关的3个重要因子核糖体大亚基蛋白L11、多肽链释放因子eRF1和eRF3在八肋游仆虫细胞中进行了荧光共定位分析。结果显示,在八肋游仆虫细胞中,蛋白质翻译过程主要位于"C"形大核内侧区域。构建的人工染色体能够作为一种有效的工具,对目的蛋白质在八肋游仆虫细胞中进行定位分析。     

游仆虫肽链释放因子eRF1对编程性翻译移码的影响

编程性翻译移码是mRNA翻译为多肽链时核糖体沿mRNA正向或反向滑动1个碱基才能表达出1个完整多肽链的现象. 人的肽链释放因子eRF1对HIV-1病毒的编程性-1移码有直接的影响. 而且在频繁发生编程性+1移码的单细胞真核生物游仆虫中,肽链释放因子eRF1对编程性移码也有明显的影响. 为进一步研究eRF1中影响编程性翻译移码的关键序列及调控机理,本研究将含有不同终止密码子的移码序列和已报道的游仆虫移码基因Ndr2分别插入双荧光素酶报告基因中,成功建立了可在酵母中进行研究的编程性移码报告检测体系. 利用游仆虫肽链释放因子Eo-eRF1b的N结构域和酵母肽链释放因子Sc eRF1的MC结构域构建了杂合肽链释放因子(Eo/Sc eRF1),检测Eo-eRF1b N结构域中的不同突变位点对移码效率的影响. 结果表明,游仆虫肽链释放因子eRF1b中YCF区的突变能明显促进含终止密码UAA的移码序列的移码,推测这可能是由于eRF1突变体降低了对UAA的识别所导致. 此外,杂合肽链释放因子Eo/Sc eRF1能够有效地提高移码基因Ndr2的移码效率. eRF1b中YCF区的突变同样能明显促进 Ndr2的移码. 因此, 游仆虫肽链释放因子YCF区的特殊序列可能是这种生物中发生编程性移码频率较高的原因之一. 本研究为探讨纤毛虫编程性翻译移码调控机制提供了实验数据.     

八肋游仆虫两类释放因子的相互作用

从八肋游仆虫中克隆到两类释放因子基因Eo-eRFI和Eo-eRF3。在Eo-eRF3基因的阅读框中有3个通用的终止密码子UGA,在此编码半胱氨酸。为了研究两类释放因子的相互作用,用PCR的方法对3个位点进行了定点突变,将UGA突变为通用的编码半胱氨酸的密码子UGU。突变结果经测序确认后,在大肠杆菌中获得全长Eo-eRF3的正确表达。在此基础上,构建酵母双杂交重组质粒,用该系统检测了游仆虫两类释放因子的相互作用。结果显示,两类释放因子在生物体内形成复合体,从而在较原始的真核生物中,证实了两类释放因子的相互作用关系。     

人eRF3a与survivin相互作用的结构域分析

第二类肽链释放因子eRF3(eukaryotic polypeptide release factor)是一种GTPase,它促进第一类肽链释放因子eRF1的释放活性,并与细胞周期调控、细胞骨架组装、细胞凋亡和肿瘤形成等过程相关。哺乳动物细胞中eRF3有两种——eRF3a和eRF3b,分别由GSPT1和GSPT2(G1 to S phase transition 1/2)基因编码。生存素(survivin)是迄今发现的最强有力的凋亡抑制因子,具有独特的结构和复杂的功能,不仅可以抑制细胞凋亡,还参与细胞有丝分裂、血管的生成等过程。eRF3和survivin都与细胞周期和细胞凋亡的调控相关。该实验室的前期研究表明,eRF3和survivin具有相互作用关系。该研究进一步对eRF3a进行截短突变,采用酵母双杂交和pull-down两种分析方法依次验证eRF3a(1-72aa)和eRF3a(1-36aa)与survivin的相互作用关系。结果表明,eRF3a(1-72aa)和eRF3a(1-36aa)均可以与survivin相互作用,由此确定eRF3a与survivin相互作用的最小结构域位于其N末端1-36aa之间,从而为进一步证实eRF3a的N端结构域与survivin协同作用参与细胞周期和细胞凋亡的调控提供了数据支持。     

Cloning, characterization and expression of the polypeptide release factor gene, eRF1, of Blepharisma japonicum

The polypeptide release factor gene, eRF1, of Blepharisma japonicum (Bj-eRF1) was cloned and sequenced. Its coding region was 1314 base pairs and encodes a protein of 437 amino acids. The cloned gene was expressed in Escherichia coli and the recombinant Bj-eRF1 polypeptide was purified by Ni2+-nitrilotriacetic acid agarose and Superose12 chromatography. Pull-down analysis showed that the recombinant Bj-eRF1 interacts with the heterologously-expressed release factor, eRF3C, of Euplotes octocarinatus.     

Nuclear localization of eukaryotic class II release factor (eRF3): implication for the multifunction of eRF3 in ciliates Euplotes cell

Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis.     

The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1

Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.     

Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.     

C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids.

Translation termination in eukaryotes requires a stop codon-responsive (class-I) release factor, eRF1, and a guanine nucleotide-responsive (class-II) release factor, eRF3. Schizosaccharomyces pombe eRF3 has an N-terminal polypeptide similar in size to the prion-like domain of Saccharomyces cerevisiae eRF3 in addition to the EF-1alpha-like catalytic domain. By in vivo two-hybrid assay as well as by an in vitro pull-down analysis using purified proteins of S. pombe as well as of S. cerevisiae, eRF1 bound to the C-terminal one-third domain of eRF3, named eRF3C, but not to the N-terminal two-thirds, which was inconsistent with the previous report by Paushkin et al. (1997, Mol Cell Biol 17:2798-2805). The activity of S. pombe eRF3 in eRF1 binding was affected by Ala substitutions for the C-terminal residues conserved not only in eRF3s but also in elongation factors EF-Tu and EF-1alpha. These single mutational defects in the eRF1-eRF3 interaction became evident when either truncated protein eRF3C or C-terminally altered eRF1 proteins were used for the authentic protein, providing further support for the presence of a C-terminal interaction. Given that eRF3 is an EF-Tu/EF-1alpha homolog required for translation termination, the apparent dispensability of the N-terminal domain of eRF3 for binding to eRF1 is in contrast to importance, direct or indirect, in EF-Tu/EF-1alpha for binding to aminoacyl-tRNA, although both eRF3 and EF-Tu/EF-1alpha share some common amino acids for binding to eRF1 and aminoacyl-tRNA, respectively. These differences probably reflect the independence of eRF1 binding in relation to the G-domain function of eRF3 (i.e., probably uncoupled with GTP hydrolysis), whereas aminoacyl-tRNA binding depends on that of EF-Tu/EF-1alpha(i.e., coupled with GTP hydrolysis), which sheds some light on the mechanism of eRF3 function.     

GTPase activity analysis of eRF3 in Euplotes octocarinatus

In eukaryotes, eRF3 participates translation termination and belongs to the superfamily of GTPase. In this work, dissociation constants for E. octocarinatus eRF3 binding to nucleosides in presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, the GTP hydrolyzing assay of Eo-eRF3 was carried out by HPLC methods and the kinetic parameter for GTP hydrolysis by eRF3 was determined. The results showed eRF1a could promote GTP binding to eRF3 and hydrolyzing GTP activity of eRF3. The observation is consistent with the data from human. Whereas E. octocarinatus eRF3 alone can bind GTP in contrast to no GTP binding observed in the absence of eRF1 in human eRF3. The affinity for Eo-eRF3 binding nucleotides is different from that in human. Structure model and amino acids sequence alignment of potential G domains indicated these different may be due to Valine 317 and Glutamate 452 displacing conserved Glycine and Lysine, which were involved in GTP binding.