同义密码子通过精微翻译选择机制实现对基因的表达调控

自然界中,同义密码子的存在使得众多氨基酸能够同时被多种密码子编码合成。随着研究的深入,同义密码子使用偏嗜性发挥出的生物学功能已经渗透到了基因复制、转录、翻译以及化学修饰等生命活动过程中。基于同义密码子使用偏嗜性的生物学特性,陆续发现密码子对(codon pair)和密码子共现(codon co-occurrence)同样在使用模式上存在明显的偏嗜性。在基因表达的过程中,针对编码序列的密码子优化能够显著提升基因的表达水平,这在生物工程领域对于蛋白表达有着重要的生物学意义。此外,同义密码子使用模式在调控基因转录、化学修饰以及翻译过程中间接控制着细胞内生命活动的有序性。而这些与同义密码子使用模式有着千丝万缕联系的生命过程主要是受精微翻译选择压力来调控运行的。本文中,我们结合当前同义密码子使用模式介导的精微翻译选择压力,简述密码子使用模式如何从转录、化学修饰以及翻译等方面来影响基因表达及蛋白产物生物学功能。这将为今后生物工程学领域如何优化蛋白高效表达以及深入研究重要生物学活动中基因表达调控提供可参考的思路与理念。     

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。     

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。     

密码子中的密码:密码子偏好性与基因表达的精细调控

同义突变由于不改变编码蛋白质的氨基酸序列,常被认为是"沉默"突变.实际上,同义密码子的选择在进化尺度上是受到限制的,从而致使同义密码子的使用频率存在差异,称为密码子偏好性.密码子偏好性在转录、转录后加工、mRNA稳定性、翻译起始、延伸、蛋白折叠等方面都起着精细调节的作用.因此,同义突变在很多情况下可导致癌症等各类疾病的发生.本综述在分子机制层面简述了近年来关于密码子偏好性对翻译和转录过程调节作用的进展,以及对于基础研究及医学方面的意义.     

密码子偏性及其应用

密码子偏性(codon bias)是指生物体中同义密码子非均衡使用的现象.研究密码子偏性可以使人们更好地理解分子进化、翻译调控等生命过程.在介绍密码子偏性的基础上,概述了影响其形成的各生物学基础,并指出其应用和发展前景.     

非编码蛋白RNA的遗传调控

大规模cDNA文库的测序和Tiling基因芯片研究结果表明, 人类基因组中大约50%的DNA可以转录为RNA, 其中只有2%能够翻译蛋白质(即mRNA), 其余98%为非编码蛋白RNAs(ncRNAs). 最近研究初步显示, 这些ncRNAs可以通过多种遗传机理调控DNA的结构、RNA的表达及蛋白质的翻译和功能, 进而在细胞、组织或个体水平上影响生物体的正常生长发育. 迄今, 人们仍对这占转录RNA 95%以上的ncRNA的功能了解甚少, 但弄清这些ncRNAs在遗传信息传递过程中的作用机理和个体发育过程中的生物功能, 是揭示生命奥妙不可缺少的环节. 为促进当今生命科学中这个最活跃研究领域的进展, 特综述ncRNA的历史和现状、作用机理及功能、发现和鉴定方法, 以及功能研究手段和应用理论、方法与实例.     

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.     

mRNA的序列、结构以及翻译速率与蛋白质结构的关系

mRNA所包含的核苷酸序列通过三联体密码子决定了蛋白质的氨基酸序列。但是, 由于对氨基酸同义密码使用频率上的差异, 密码子与反密码子相互作用效率上的不同, 以及密码子上下文关系和mRNA 不同区域二级结构上的差异, 造成了核糖体对mRNA 不同区域翻译速度上的差异, 加之共翻译折叠的作用, 使得mRNA 的序列和结构影响着蛋白质空间结构的形成。     

P-body及其与mRNA的转录后调节之间的关系

mRNA在真核生物基因表达的转录后调控中发挥着重要作用.然而最新研究发现,一种被命名为P-body胞浆复合体是mRNA转录后调控过程中的一个重要场所,在基因表达过程中起到了至关重要的调控作用.该复合体富含多种功能的蛋白,同时还有细胞因子与RNA等成分组成,其功能特征为参与mRNA降解、翻译抑制、mRNA监视以及RNA介导基因沉默等重要生命活动过程.研究发现,P-body是特化的细胞成分.本文详细阐述P-body的发现、结构特征以及与mRNA转录后调控的关系,使人们对P-body的生物学功能有更深入地了解和认识,另一方面将有助于更进一步深入探讨基因的表达调节机制..     

mRNA翻译起始区二级结构改变对干扰素基因在大肠杆菌中翻译的影响

为研究mRNA翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变α8干扰素及αA干扰素衍生物基因的5′端若干位点,使其与表达载体重组后转录形成的mRNA翻译起始区结构发生改变。SDS-PAGE及活性测定证实这些改变提高了外源基因的表达水平。RNA斑点印迹表明突变前后基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高。mRNA翻译起始区二级结构预测提示其生成自由能(ΔG)的变化可能与表达水平的提高有关。     

The translation factors of Drosophila melanogaster

Synthesis of polypeptides from mRNA (translation) is a fundamental cellular process that is coordinated and catalyzed by a set of canonical ‘translation factors’. Surprisingly, the translation factors of Drosophila melanogaster have not yet been systematically identified, leading to inconsistencies in their nomenclature and shortcomings in functional (Gene Ontology, GO) annotations. Here, we describe the complete set of translation factors in D. melanogaster, applying nomenclature already in widespread use in other species, and revising their functional annotation. The collection comprises 43 initiation factors, 12 elongation factors, 3 release factors and 6 recycling factors, totaling 64 of which 55 are cytoplasmic and 9 are mitochondrial. We also provide an overview of notable findings and particular insights derived from Drosophila about these factors. This catalog, together with the incorporation of the improved nomenclature and GO annotation into FlyBase, will greatly facilitate access to information about the functional roles of these important proteins.     

GTPases of the Translation Apparatus

Protein biosynthesis is a complex biochemical process involving a number of stages at which different translation factors specifically interact with ribosome. Some of these factors belong to GTP-binding proteins, or G-proteins. Due to their functioning, GTP is hydrolyzed to yield GDP and the inorganic phosphate ion P_i. Interaction with ribosome enhances GTPase activity of translation factors; i.e., ribosome plays a role of GTPase-activating protein (GAP). GTPases involved in translation interact with ribosome at every stage of protein biosynthesis. Initiation factor 2 (IF2) catalyzes initiator tRNA binding to the ribosome P site and subsequent binding of the 50S subunit to the initiation complex of the 30S subunit. Elongation factor Tu (EF-Tu) controls aminoacyl-tRNA delivery to the ribosome A site, while elongation factor G (EF-G) catalyzes translocation of the mRNA-tRNA complex by one codon on the ribosome. Release factor 3 (RF3) catalyzes the release of termination factors 1 or 2 (RF1 or RF2) from the ribosomal complex after completion of protein synthesis and peptidyl-tRNA hydrolysis. The functional properties of translational GTPases as related to other G-proteins, the putative mechanism of GTP hydrolysis, structural features, and the functional cycles of translational GTPases are considered.     

核糖体谱技术及其应用

当前,基因组范围内分析细胞内蛋白质翻译事件的研究仍然落后于转录事件的研究.无论原核生物还是真核生物,细胞内的核糖体均是蛋白质翻译的工厂,因此对于核糖体的研究至关重要.早在1963年,Warner等[1]发现了细胞内的多聚核糖体结构.Wolin和Walter[2]用RNase消化正在翻译中     

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation I_TE is formulated to facilitate studies on the efficiency and evolution of the translation machinery.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

A snapshot of translation in Mycobacterium tuberculosis during exponential growth and nutrient starvation revealed by ribosome profiling

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The ecology of the free-living stages of Ostertagia circumcincta

Callinan A. P. L. 1978. The ecology of the free-living stages of Ostertagia circumcincta. Internationaljournal for Parasitology8: 233–237. The development and survival of the free-living stages O. circumcincta was studied in western Victoria in 1974–1976. For all plots in which development occurred, pre-infective larvae (L₁-L₂) were recovered within 0–8 days, infective larvae (L₃) within 7–30 days and L₃ on herbage and soil within 4–27 days. The mean minimum development time to L₃ on herbage and soil was 14·3 ± 3·5 days and the mean development time to maximum yield of these was 33·3 ± 6·3 days. There were no distinct differences in development rates between plots. Plot yields of L₃ on herbage and soil varied inversely as the mean of daily temperatures for the period until maximum yield of these. Yields varied from 0 to 16% of the number of eggs put out on each plot and highest yields were obtained from eggs put out during late autumn-winter. No L₃ were observed to survive over summer. The migratory habits of L₃ were such that a mean of 75·1 ± 5·6% of L₃ on herbage and soil were actually on herbage; the soil was always a significant source of L₃.     

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.