tRNA在基因表达中的调控作用

转移核糖核酸（transfer RNA,tRNA）是遗传信息传递过程中的“适配器”分子,它们能够把mRNA所携带的遗传信息准确地翻译成蛋白质的氨基酸序列.然而,近年来的研究表明,tRNA在基因表达过程中还具有重要的调控作用.当生物面临外界某些营养压力胁迫时,空载tRNA可作为效应分子影响细胞整体基因表达水平,从而使机体应对不利的环境.在酵母和某些哺乳动物细胞中,tRNA可以从细胞质逆行回细胞核.这种逆行一方面可以使细胞核的监控系统连续地监控tRNA的完整性,另一方面,在营养缺乏时,逆行回细胞核的tRNA可以有效地降低蛋白质的合成水平.最新研究表明,tRNA并不是绝对稳定的RNA分子.在某些生理或逆境胁迫下,tRNA在其反密码环或反密码环左臂处被内切酶特异性切割成不同长度的片段.这些切割并不是无意义的随机降解现象,而有可能产生一类新的信号分子,如tRNA半分子或sitRNA,它们与生物应激反应中细胞整体代谢的基因表达调控有着密切的关系.关于tRNA调控功能的研究是一个新的前沿领域,它将揭示tRNA结构与功能的多样性及其在遗传信息表达过程中的重要作用.     

毕赤酵母中tRNAPro CCG基因的表达及其作用效果

转运核糖核酸 (tRNA) 是蛋白质合成过程中重要参与成分之一,为了探索稀有密码子对应的tRNA (稀少tRNA) 丰度改变对外源基因表达量的影响,文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区,结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNAPro CCG基因顺次连接于pPIC9K载体上,在毕赤酵母GS115中共表达,结果使GFP表达量提高了4.9%;另将带有阻遏区的GFP基因和tRNAPro CCG基因分别连接于pPIC9K和pFLDα载体,在毕赤酵母GS115中共表达,GFP表达量最高提高了12.5%;应用同样方式将tRNAPro CCG基因与NFATc3T-GFP融合基因共表达,其表达量提高了21.3%。可见,tRNAPro CCG在毕赤酵母GS115中确为稀少tRNA,通过共表达tRNAPro CCG基因可显著提高带有连续该密码子的外源基因表达量,并且,文中构建的共表达体系将同样适用于其他稀少tRNA基因的筛选和验证。     

转移核糖核酸(tRNA)与癌症

转移核糖核酸(transfer RNA, tRNA)在蛋白质生物合成过程中起关键作用,是将遗传信息翻译成蛋白质一级结构的接头分子。tRNA长久以来一直被认为是基因表达调控过程中的执行者而不具备调控功能,更不曾与癌症的发生联系起来。最新研究表明,某些tRNA在癌细胞中异常表达,与癌症的发生和发展有密切联系。tRNA来源的小分子非编码RNA (tRFs和tiRNAs)是一类新的基因表达调控分子,tRFs可以调控癌基因的表达或者与RNA结合蛋白相互作用来调控癌细胞增殖和细胞周期进程。tRNA的转录后修饰能够调控mRNA翻译过程,进而影响癌细胞的生长。随着测序技术的发展,tRNA在癌症发生和发展中的调控作用成为近年来的研究热点,现将从"tRNA分子调控癌症的发生和发展"、"tRNA来源的小分子非编码RNA与癌症"以及"tRNA修饰与癌症"三个方面综述tRNA分子在癌症发生和发展中的调控功能。     

内含子对真核基因表达调控的影响

大多数真核基因都含有非编码的间隔序列--内含子,根据剪接机制的不同,可将内含子分为3类:真核mRNA内舍子、自我剪接内含子和真核tRNA内含子.在多数情况下,真核mRNA内含子的存在可以提高基因的表达水平.因为其剪接过程会影响mRNA新陈代谢的多个阶段,包括转录、RNA编辑、pre-mRNA的加工、mRNA的出核运输、翻译和无义衰变等.真核mRNA内含子在真核生物基因表达调控中起着重要的作用,是转基因研究中提高外源基因表达的重要元件之一.就真核mRNA内含子的特性、剪接机制及其对真核基因表达调控的影响作一概述.     

线粒体基因表达的调控及其与某些疾病的关系

线粒体除作为细胞生成能量的场所,其基因组还编码参与线粒体氧化呼吸链组成的13条多肽链,22种tRNA分子和2种rRNA分子。线粒体基因表达受众多因素调控,其表达异常可影响细胞对氧的利用,与一系列病理生理过程密切相关。该文介绍近年来线粒体基因表达调控,及肿瘤等疾病过程中线粒体基因表达变化的研究进展。     

毕赤酵母中tRNA_(CCG)~(Pro)基因的表达及其作用效果

转运核糖核酸(tRNA)是蛋白质合成过程中重要参与成分之一,为了探索稀有密码子对应的tRNA(稀少tRNA)丰度改变对外源基因表达量的影响,文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区,结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNA_(CCG)~(Pro)基因顺次连接于pPIC9K载体上,在毕赤酵母GS115中共表达,结果使GFP表达量提高了4.9%;另将带有阻遏区的GFP基因和tRNA_(CCG)~(Pro)基因分别连接于pPIC9K和pFLDα载体,在毕赤酵母GS115中共表达,GFP表达量最高提高了12.5%;应用同样方式将tRNA_(CCG)~(Pro)基因与NFATc3T-GFP融合基因共表达,其表达量提高了21.3%。可见,tRNA_(CCG)~(Pro)在毕赤酵母GS115中确为稀少tRNA,通过共表达tRNA_(CCG)~(Pro)基因可显著提高带有连续该密码子的外源基因表达量,并且,文中构建的共表达体系将同样适用于其他稀少t RNA基因的筛选和验证。     

SnRNA对基因表达的调控

基因表达的调控机制研究是生物学中十分活跃的领域,基因重组技术对基因的分离和结构分析的报道不胜枚举,其目的在于阐明基因表达的调控机理;在原核生物中,已发现蛋白质作为基因表达调节因子,因此,大部分研究者注重于蛋白质分子在基因表达中的调控作用,对于DNA分子不重视,只是把它看作是基因表达过程中的过渡阶段如mRNA、tRNA、rRNA。近年来发现RNA具有酶功能、参予mRNA成熟过程中的拼接、DNA复制、染色质结构及基因表达的调控,种类繁多,已引起了生物学者的关注。     

tRNA核质动态分布与细胞命运

旺盛的细胞核、质间的物质运输(nuclear-cytoplasmic transport)是真核细胞代谢的基础．核质运输不仅将蛋白质运到目的地,还能通过在特定时间、地点结合靶分子,改变其在胞内的局部浓度,调控诸如有丝分裂等重要细胞活动．tRNA是细胞中最重要的大分子之一,合成于细胞核,在细胞质中参加蛋白质翻译．一直以来,学术界认为tRNA只是蛋白质合成的参与者,tRNA核质运输是tRNA跨越核膜进入细胞质是单向主动运输过程．然而,最近的研究成果在颠覆传统观念,tRNA不但能被转运出核,还能被逆向转运入核．2008年,新概念“tRNA核质动态分布”(tRNA nuclear-cytoplasmic dynamics)被提出,取代tRNA核质运输,描述tRNA在细胞核、质间的流动．在酿酒酵母中tRNA核质动态分布可以调控蛋白质翻译,锁定细胞周期．此领域内的最新研究正在改变着教科书中有关tRNA的传统论断．     

tRNA核苷修饰与细胞胁迫应答

细胞的生长和功能发挥需要特定的内部条件。当外界条件发生变化时,细胞要想保持这种特定的内部环境,需要许多过程的参与,其中最重要的一个部分是RNA代谢调节,其通常涉及一般翻译水平的下降和应激反应,以有利基因翻译的增加。tRNA是翻译机制的一个基本组成部分,在蛋白质合成过程中,它将氨基酸传递给核糖体。tRNA的显著特征之一是高度修饰,这些修饰有大量用途,包括确保翻译的准确性和高效性、维持tRNA折叠或稳定性等。细胞在逆境胁迫条件下,tRNA修饰水平会发生显著变化,并通过不同的途径影响细胞的翻译。本文阐述了tRNA核苷修饰与细胞胁迫之间的相互关系,描述了tRNA修饰响应胁迫应答的可能机制。     

真核细胞tRNA的降解

tRNA在蛋白质合成过程中起着关键性的作用,不但为三联密码子翻译成氨基酸提供了接合体,而且为将氨基酸运送到核糖体提供了运送载体.在真核细胞中,tRNA前体必须经过广泛的加工修饰,成为成熟的tRNA分子才能充分发挥生物学功能.以往对tRNA的研究主要集中于tRNA的结构、功能、加工和成熟上,却很少关注tRNA分子的降解.最近研究发现tRNA的降解在tRNA的生成、加工和功能发挥上同样起着重要作用.     

嗜酸热硫化叶菌麦芽寡粉基海藻糖合酶基因的克隆和表达

The gene of MTSase (maltooligosyltrehalose synthase) from Sulfolobus acidocaldarius ATCC49426 was amplified by PCR. The primers were designed according to the published sequence of homologous gene from Sulfolobus acidocaldarius ATCC33909. This gene was inserted into the plasmid pBV220 and the resultant recombinant plasmid pBV220-GT was transformed to E. coli DH5 alpha. The activity of recombinant enzyme was about 10 u/g(wet cell). In order to improve the expression level of target protein, some nucleotides in the 3' and 5' of the gene were modified to optimize the second structure of mRNA by PCR amplification using the new primers devised according to the biosoftware GOLDKEY2.0. As a result, the activity of recombinant enzyme increase to 19.8 u/g(wet cell). Then, the helping plasmid pUBS520 which carried the gene encoding the tRNA of rare codons AGG and AGA was transformed to the recombinant strain. But it took little effect.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.     

A functional link between housekeeping selenoproteins and phase II enzymes

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.     

A role for tRNA modifications in genome structure and codon usage

Transfer RNA (tRNA) gene content is a differentiating feature of genomes that contributes to the efficiency of the translational apparatus, but the principles shaping tRNA gene copy number and codon composition are poorly understood. Here, we report that the emergence of two specific tRNA modifications shaped the structure and composition of all extant genomes. Through the analysis of more than 500 genomes, we identify two kingdom-specific tRNA modifications as major contributors that separated archaeal, bacterial, and eukaryal genomes in terms of their tRNA gene composition. We show that, contrary to prior observations, genomic codon usage and tRNA gene frequencies correlate in all kingdoms if these two modifications are taken into account and that presence or absence of these modifications explains patterns of gene expression observed in previous studies. Finally, we experimentally demonstrate that human gene expression levels correlate well with genomic codon composition if these identified modifications are considered.