同义密码子使用模式对多肽链共翻译折叠的影响研究进展

同义密码子使用模式作为核苷酸与氨基酸的纽带,其多样性介导了核糖体扫描速率,同时扩充了基因的遗传信息存储量。随着新型技术的应用,发现特异性密码子和密码子结合力可调节核糖体扫描速率并影响蛋白质构象。同义密码子使用模式通过多种方式在不同环节影响着核糖体扫描速率,同时还影响着自身mRNA的稳定性。本文简述了密码子使用模式如何在核糖体扫描翻译mRNA的过程中实现对多肽链翻译延伸的调控,为今后生物工程学领域如何优化蛋白高效表达提供可参考的思路与理念。     

哺乳动物线粒体蛋白质翻译及其调控机制

线粒体是真核细胞内参与能量生成和物质代谢的重要细胞器。线粒体核糖体(mitochondrial ribosome, MR)作为细胞器中的翻译机器,用于表达线粒体DNA(mitochondrial DNA, mtDNA)编码的基因。近年来,随着研究的不断深入,人们对参与哺乳动物线粒体蛋白质翻译的蛋白质因子及其翻译的基本过程有了越来越清晰的认识,这对阐明线粒体蛋白质翻译的调控机制及研究人类线粒体疾病等方面具有重要的意义。线粒体蛋白质的翻译过程分为起始、延伸、终止和回收四个阶段。本文综述哺乳动物线粒体核糖体的结构与功能,以及线粒体蛋白质翻译因子的性质与功能,并进一步探讨翻译激活因子、微小RNA、线粒体COX翻译调控组装中间体(mt-translation regulation assembly intermediate of COX, MITRAC)以及核糖体的翻译后修饰对线粒体蛋白质翻译的调控及其机制,展望其对人类线粒体相关疾病研究的应用前景。     

枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响

用枯草杆菌体外转录-翻译偶联系统检测13种19株枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响,发现10种13株核糖体蛋白质突变能影响碱性蛋白酶基因的表达。其中依赖链霉素突变核糖体几乎不能翻译碱性蛋白酶mRNA。依赖链霉素突变在翻译层次抑制碱性蛋白酶基因的表达,但对中性蛋白酶基因的表达没有影响。在碱性蛋白酶mRNA翻译起始区有一个复合二级结构,用体外突变方法破坏其中一个,翻译效率提高8.2倍。依赖链霉素突变和抗链霉素突变核糖体的高级结构不同,与碱性蛋白酶mRNA 5'端片段的亲合力也有差异。由于碱性蛋白酶mRNA翻译起始区的复合二级结构和低起始强度以及依赖链霉素突变核糖体高级结构的改变,使依赖链霉素突变核糖体不能翻译碱性蛋白酶mRNA。     

单分子荧光共振能量转移技术在核糖体移位研究中的进展

核糖体是蛋白质的"合成工厂",也是临床上多种抗菌药物的作用靶点,因此,深入理解细菌核糖体的蛋白质翻译机制意义重大.蛋白质翻译是通过多步骤相互协调、多组分精细配合来实现高保真和精确调控.核糖体在mRNA上的移位作为翻译过程中最重要的事件之一,需要核糖体大规模的构象重排以及tRNA2-mRNA沿着核糖体的精确移动.在细菌中,移位是由延伸因子EF-G催化GTP水解来驱动的.近年来,单分子荧光共振能量技术(smFRET)的发展使得人们可以探究单个tRNA分子移位的动力学过程并实时观测核糖体的构象变化.本文首先介绍了smFRET技术的原理及特点,对其在核糖体结构动态及tRNA移位研究中的应用进行了较为系统的总结,并对其应用前景进行了展望.     

同义密码子使用模式对蛋白产物表达及构象形成的影响*

鉴于遗传密码子的简并性能够将基因遗传信息的容量提升,同义密码子使用偏嗜性得以在生物体的基因组中广泛存在。虽然同义密码子之间碱基的变化并不能导致氨基酸种类的改变,在研究mRNA半衰期、编码多肽翻译效率及肽链空间构象正确折叠的准确性和翻译等这一系列过程中发现,同义密码子使用的偏嗜性在某种程度上通过精微调控翻译机制体现其遗传学功能。同义密码子指导tRNA在翻译过程中识别核糖体的速率变化是由氨基酸的特定顺序决定,并且在新生多肽链合成时,蛋白质共翻译转运机制同时调节其空间构象的正确折叠从而保证蛋白的正常生物学功能。某些同义密码子使用偏嗜性与特定蛋白结构的形成具有显著相关性,密码子使用偏嗜性一旦改变将可能导致新生多肽空间构象出现错误折叠。结合近些年来国内外在此领域的研究成果,阐述同义密码子使用偏嗜性如何发挥精微调控翻译的生物学功能与作用。     

线粒体核糖体蛋白质与人类恶性肿瘤

线粒体拥有自身独特的核糖体--线粒体核糖体,用于翻译线粒体DNA（mitochondrial DNA, mtDNA）编码的基因。线粒体核糖体由核基因编码的线粒体核糖体蛋白质(mitochondrial ribosomal protein, MRPs)和线粒体自身编码的rRNA组装而成。MRPs表达失调会引发代谢紊乱、呼吸链受损,导致细胞发生功能障碍和异常增殖,甚至发生癌变等恶性转化。大量研究证明,MRPs在不同的肿瘤细胞中表达异常,提示着MRPs在肿瘤发生发展过程中发挥着重要作用。本文就线粒体核糖体蛋白质与人类恶性肿瘤发生的关系作一综述,为进一步阐明其在恶性肿瘤发生过程中的作用机制奠定基础。     

mRNA 5'端发卡结构与翻译的起始调控

mRNA的5’端大多存在各种由RNA自身折叠形成的发卡结构,这些结构对于核糖体识别mRNA翻译起始位点并与之准确结合往往具有十分重要的意义。简要综述了此类结构的基本特征以及它们在生物体内所起到的调控蛋白质翻译的作用。     

交联法在分子生物学研究中的应用

本文概要介绍了交联法用于阐明染色质、核小体、病毒粒子、核糖体结构、观察HS70基因-组蛋白等七种核酸-蛋白质复合物中大分子相互作用的实例;列举了它在DNA构象、RNA立体结构及mRNA、tRNA、snRNA、hnRNA、rRNA间相互作用等研宄中的应用。从文中可以看出,交联法的应用范围遍及遗传信息储存的高级结构、转录体系及调控、翻译系统及调控等分子生物学研究的几个主要侧面。     

真核细胞中多重表达框mRNA的选择性翻译起始

扫描模型和遗漏扫描模型是真核生物mRNA翻译起始的两种主要机制,但其仍存在某些例外情况,如对具有多顺反子结构的mRNA,选择性翻译起始的发生机制目前仍不清楚.本研究基于GFP蛋白开放表达框(ORF)构建了一系列重组表达载体,用以转录在移码翻译顺序及同一翻译顺序下,AUG起始密码子处于不同序列背景,以及间隔不同距离的多顺反子结构mRNA.通过转染人Bel 7402细胞系,研究了这些多顺反子结构mRNA的翻译起始模式.结果表明,在移码翻译顺序下,多顺反子mRNA可翻译出对应的不同蛋白质,而在同一翻译顺序下,GFP蛋白表达框中的多个AUG密码子,仅有首位起始密码子可发挥作用,提示核糖体在从首位起始密码子开始翻译的同时,可能会有部分核糖体继续向下扫描并识别下游的起始密码子,而这种选择性的翻译起始效率,主要取决于密码子所处的序列背景及间隔距离等因素.     

[D丙~6,脯~9乙基胺]-促黄体素释放激素LH-RHA对妊娠兔子宫内膜多聚核糖体mRNA的作用

本实验制备了非孕兔、孕兔和绐孕兔注射高剂量[D丙‘,脯’乙基胺]-促黄体素释放激素(LH-RH A)不同天数的子宫内膜多聚核糖体,并从多聚核糖体提取mRNA,在网织红细胞无细胞翻译系统中测定了活性。结果指出用LH-RH A处理后多聚核糖体mRNA量减少,其翻译活性降低,在体内实验中核糖体mRNA诱导兔子宫分秘蛋白的合成也受到抑制,特别是分子量大约为22,000和69,000左右的分泌蛋白合成受到明显抑制。     

Functional compatibility of elongation factors between mammalian mitochondrial and bacterial ribosomes: characterization of GTPase activity and translation elongation by hybrid ribosomes bearing heterologous L7/12 proteins

The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.     

A structural model for the large subunit of the mammalian mitochondrial ribosome

Protein translation is essential for all forms of life and is conducted by a macromolecular complex, the ribosome. Evolutionary changes in protein and RNA sequences can affect the 3D organization of structural features in ribosomes in different species. The most dramatic changes occur in animal mitochondria, whose genomes have been reduced and altered significantly. The RNA component of the mitochondrial ribosome (mitoribosome) is reduced in size, with a compensatory increase in protein content. Until recently, it was unclear how these changes affect the 3D structure of the mitoribosome. Here, we present a structural model of the large subunit of the mammalian mitoribosome developed by combining molecular modeling techniques with cryo-electron microscopic data at 12.1A resolution. The model contains 93% of the mitochondrial rRNA sequence and 16 mitochondrial ribosomal proteins in the large subunit of the mitoribosome. Despite the smaller mitochondrial rRNA, the spatial positions of RNA domains known to be involved directly in protein synthesis are essentially the same as in bacterial and archaeal ribosomes. However, the dramatic reduction in rRNA content necessitates evolution of unique structural features to maintain connectivity between RNA domains. The smaller rRNA sequence also limits the likelihood of tRNA binding at the E-site of the mitoribosome, and correlates with the reduced size of D-loops and T-loops in some animal mitochondrial tRNAs, suggesting co-evolution of mitochondrial rRNA and tRNA structures.     

Proteomic analysis of the mammalian mitochondrial ribosome. Identification of protein components in the 28 S small subunit

The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M., Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, K. (2001) J. Biol. Chem. 276, 21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify cDNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.     

Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome. Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria

The mammalian mitochondrial ribosome (mitoribosome) is a highly protein-rich particle in which almost half of the rRNA contained in the bacterial ribosome is replaced with proteins. It is known that mitochondrial translation factors can function on both mitochondrial and Escherichia coli ribosomes, indicating that protein components in the mitoribosome compensate the reduced rRNA chain to make a bacteria-type ribosome. To elucidate the molecular basis of this compensation, we analyzed bovine mitoribosomal large subunit proteins; 31 proteins were identified including 15 newly identified proteins with their cDNA sequences from human and mouse. The results showed that the proteins with binding sites on rRNA shortened or lost in the mitoribosome were enlarged when compared with the E. coli counterparts; this suggests the structural compensation of the rRNA deficit by the enlarged proteins in the mitoribosome.     

mim3 and nam3 omnipotent suppressor genes similarly affect the polypeptide composition of yeast mitoribosomes

Yeast informational suppressors of mit- mutations coded for by nuclear (nam3-1, nam3-2) or by mitochondrial DNA (mim3-1) affect the mitoribosome. Nuclear mutations result in the appearance of an additional polypeptide called SI in the small mitoribosomal subunit. An identical polypeptide, not detected in the wild type 37S subunit, is present in crude preparations of mitoribosomes isolated from a mim3-1 suppressor carrying strain. Traces of the SI polypeptide may be found in highly purified small subunits from the mim3-1 strain. Therefore, mutations affecting either mitochondrial rRNA (mim3-1) or mitochondrial r-proteins (nam3-1, nam3-2) could be followed by similar changes in overall mitoribosome structure. This may explain the functional similarity of nuclear and mitochondrially coded suppressors.     

Insights into mitoribosomal biogenesis from recent structural studies

The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.     

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.     

Solution Structure of the Catalytic Domain of the Mitochondrial Protein ICT1 That Is Essential for Cell Vitality

The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a β1-β2-α1-β3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.