蛋白质合成肽链释放因子的多功能性

肽链释放因子在蛋白质合成终止过程中,对新生肽链从核糖体上释放起重要作用。第一类肽链释放因子识别终止密码子,水解肽酰-tRNA酯键;第二类肽链释放因子是一类依赖于第一类肽链释放因子和核糖体的GTP酶,促进第一类肽链释放因子发挥肽链释放的功能。最近的研究表明,肽链释放因子不仅在细胞内蛋白质合成终止过程中起重要的作用,其在细胞的骨架形成,尤其是细胞有丝分裂过程中对纺锤体的形成起重要的作用。两类肽链释放因子还与其他功能蛋白质相互作用,表现出多功能蛋白质的特征。     

肽链释放因子识别终止密码子的机制

肽链释放因子(polypeptide release factor, RF)是参与细胞内蛋白质合成终止过程中新生肽链释放的一组重要的蛋白质,包括两类,即第一类肽链释放因子(classⅠrelease factor, RFⅠ)和第二类肽链释放因子(classⅡrelease factor, RFⅡ).关于第一类肽链释放因子识别终止密码子的机制和功能位点是目前分子细胞生物学领域的一个研究热点,第二类肽链释放因子作为一类GTP酶,在第一类肽链释放因子识别终止密码子和肽链释放过程中的协同作用也备受关注.近些年来,通过构建体内和体外的测活体系,对第一类肽链释放因子识别终止密码子的机制的研究取得了一些进展,提出了多种假说和模型,尤其是对第一类肽链释放因子的晶体结构及两类肽链释放因子复合体的空间结构的研究,为揭示真核生物细胞内蛋白质合成终止机制提供了直接的证据.     

第一类肽链释放因子结构与功能研究的新进展

蛋白质生物合成过程的终止是由于第一类肽链释放因子识别终止密码子,并导致肽酰-tRNA酯键水解,释放出新合成的多肽链．近期,通过冷冻电镜、结晶学、核磁共振、分子动力学和生物化学等方面的研究,使第一类肽链释放因子的结构与功能逐渐清晰．对近期的研究进行了分析和整理．     

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.     

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.     

核糖体肽链输出通道对基因表达的调控作用

核糖体大亚基上的肽链输出通道起始于肽酰转移酶中心附近,横穿大亚基,主要由rRNA和少量蛋白质组成,是新生肽链离开大亚基的通道,近年研究发现这是通道的一个动态的结构,它不仅能够与新生肽链中的效应模体相互作用翻译效率,而且能够调节通过通道的多肽链的共翻译折叠,修饰,还可协助它们在细胞中定位。     

蛋白质的结构转换

许多不相关的蛋白质含有相同的短肽序列却形成不同的空间构象. 结构转换广泛存在于蛋白质折叠和功能过程中, 具有重要的生物学意义. 综述了Serpin和EF-Tu的失活、血细胞凝集素的激活、蛋白酶成熟、亚基装配和蛋白质淀粉样化等过程中肽链同源肽段的结构转换模式, 并讨论了它在理解蛋白质折叠机理和“构象病”病因中的应用.     

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     

促肾上腺皮质激素释放因子和促肾上腺皮质激素

神经肽类激素的发现扩大了激素生理、生化研究范畴。1955年Schally和Gillemine同时发现下丘脑存在促肾上腺皮质激素释放因子(CRT)。在此发现的启示下,下丘脑九种肽类激素被发现和神经内分泌器官得到确认。然而由于它的纯化和化学结构的鉴定相当困难,有一个时期关于它的研究几乎处于停滞状态。1981年纯化的促肾上腺皮质激素释放因子的化学结构的发现为研究它的性质、功能起了巨大的推动作用。促肾上腺皮质激素释放因子和促肾上腺皮质激素(ACTH)二者之间的研究密切相关,本文试对二者的研究、进展作一综述。     

The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.

Translation termination in eukaryotes requires a codon-specific (class-I) release factor, eRF1, and a GTP/GDP-dependent (class-II) release factor, eRF3. The model of "molecular mimicry between release factors and tRNA" predicts that eRF1 mimics tRNA to read the stop codon and that eRF3 mimics elongation factor EF-Tu to bring eRF1 to the A site of the ribosome for termination of protein synthesis. In this study, we set up three systems, in vitro affinity binding, a yeast two-hybrid system, and in vitro competition assay, to determine the eRF3-binding site of eRF1 using the fission yeast Schizosaccharomyces pombe proteins and creating systematic deletions in eRF1. The in vitro affinity binding experiments demonstrated that the predicted tRNA-mimicry truncation of eRF1 (Sup45) forms a stable complex with eRF3 (Sup35). All three test systems revealed that the most critical binding site is located at the C-terminal region of eRF1, which is conserved among eukaryotic eRF1s and rich in acidic amino acids. To our surprise, however, the C-terminal deletion eRF1 seems to be sufficient for cell viability in spite of the severe defect in eRF3 binding when expressed in a temperature-sensitive sup45 mutant of the budding yeast, Saccharomyces cerevisiae. These results cannot be accounted for by the simple "eRF3-EF-Tu mimicry" model, but may provide new insight into the eRF3 function for translation termination in eukaryotes.     

Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon

Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).     

Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic

Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3.     

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.     

Making sense of mimic in translation termination

The mechanism of translation termination has long been a puzzle. Recent crystallographic evidence suggests that the eukaryotic release factor (eRF1), the bacterial release factor (RF2) and the ribosome recycling factor (RRF) all mimic a tRNA structure, whereas biochemical and genetic evidence supports the idea of a tripeptide 'anticodon' in bacterial release factors RF1 and RF2. However, the suggested structural mimicry of RF2 is not in agreement with the tripeptide 'anticodon' hypothesis and, furthermore, recently determined structures using cryo-electron microscopy show that, when bound to the ribosome, RF2 has a conformation that is distinct from the RF2 crystal structure. In addition, hydroxyl-radical probings of RRF on the ribosome are not in agreement with the simple idea that RRF mimics tRNA in the ribosome A-site. All of this evidence seriously questions the simple concept of structural mimicry between proteins and RNA and, thus, leaves only functional mimicry of protein factors of translation to be investigated.     

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

The decoding release factor （RF） triggers termination of protein synthesis by functionally mimicking a tRNA to span the decoding centre and the peptidyl transferase centre （PTC） of the ribosome. Structurally, it must fit into a site crafted for a tRNA and surrounded by five other RNAs, namely the adjacent peptidyl tRNA carrying the completed polypeptide, the mRNA and the three rRNAs. This is achieved by extending a structural domain from the body of the protein that results in a critical conformational change allowing it to contact the PTC. A structural model of the bacterial termination complex with the accommodated RF shows that it makes close contact with the first, second and third bases of the stop codon in the mRNA with two separate loops of structure＂ the anticodon loop and the loop at the tip of helix orS. The anticodon loop also makes contact with the base following the stop codon that is known to strongly influence termination efficiency. It confirms the close contact of domain 3 of the protein with the key RNA structures of the PTC. The mRNA signal for termination includes sequences upstream as well as downstream of the stop codon, and this may reflect structural restrictions for specific combinations of tRNA and RF to be bound onto the ribosome together. An unbiased SELEX approach has been investigated as a tool to identify potential rRNA-binding contacts of the bacterial RF in its different binding conformations within the active centre of the ribosome.     

Mapping functionally important motifs SPF and GGQ of the decoding release factor RF2 to the Escherichia coli ribosome by hydroxyl radical footprinting. Implications for macromolecular mimicry and structural changes in RF2

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved (250)GGQ(252) motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, (205)SPF(207), were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 A only, a distance that is incompatible with the observed cleavage sites that are up to 100 A apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.     

A dynamic competition between release factor 2 and the tRNA(Sec) decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H.

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.