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.     

3′-半分子5′-末端双链结构阻止tRNA连接酶酶促反应

用定点突变技术将不同核苷酸引入酵母苯丙氨酸tRNA反密码子环32,37和38位.体外转录制备tRNA前体,其32,37和38位的核苷酸与野生型tRNA前体相应位点的核苷酸不同.用纯化的酵母tRNA内切酶和tRNA连接酶对这些tR-NA前体进行剪接加工.结果说明,这些位点的核苷酸不仅影响tRNA内切酶对tR-NA前体的酶切效率,而且3’-半分子5’-末端双链结构阻止tRNA连接酶将相应的tRNA半分子连接成整分子tRNA.     

真核生物细胞质tRNA生物合成研究的进展

tRNA是蛋白质翻译机器中的必需成分,它对细胞的生长和增殖及器官的发育起着决定性作用.tRNA生物合成包括tRNA基因的转录、转录后的加工和修饰.对tRNA生物合成的研究还包括tRNA在细胞中的运输、tRNA生物合成的质量监控及其与其他重要细胞途径（如mRNA生物合成、DNA损伤应答和细胞周期）之间的相互作用,以及tRNA生物合成在生长发育和疾病中的作用.本文主要介绍了近年来真核生物细胞质tRNA生物合成研究的一些重要进展.     

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。     

蛋白质翻译中的反转运

在蛋白质的翻译过程中,氨酰-tRNA进入核糖体,解密mRNA上的一个密码子,并带着mRNA向其5'的方向运动,直到空载的tRNA离开核糖体,整个过程tRNA在核糖体内始终沿着一个方向运动.但随着LepA(EF4)蛋白的发现和其功能的明确,tRNA在核糖体内的新运动形式--"反转运"被揭示,即tRNA带着mRNA倒退一步,向其3'的方向运动.通过对tRNA反向运动生理意义的研究,引发了对蛋白质翻译调控的深入思考.     

遗传密码子进化的阶段性

密码子的起源与进化可划分为4个阶段,即：前密码子期,tRNA形成期,原密码子与有序肽同步起源期,以及密码子进化期。不同的观点,大都能在不同时期找到自己的位置,从而是互补的。在此基础上,首次将主要理论的主旨整合为一个进化过程的理论框架,基本上协调了持续了近半个世纪的确定论和偶然论之争,并且解释了密码子设定的多态性的由来。     

tRNA来源的小片段RNA——新的基因表达调控因子

转移核糖核酸(tRNA)是蛋白质合成的关键接头分子,特异性识别信使RNA(mRNA)的密码子信息,将其接载的氨基酸基团掺入到新生多肽链中。最新研究表明,在很多物种中,在某些特定情况下,tRNA或其前体被特异性剪切产生tRNA来源的小片段RNA(tRNA-derived fragment,tRF)。这类tRF是一类新的基因表达调控因子,其发挥作用的机制多样,如某些tRF以microRNA方式抑制mRNA翻译;某些tRF作为逆转录病毒RNA基因组的逆转录引物;而某些tRF参与了前体rRNA剪切复合物的组装。此外,细胞受胁迫产生的带有多聚鸟苷酸模块的tRF则会竞争性抑制延伸因子elF4G与mRNA的结合,从而抑制蛋白质翻译。随着研究的继续深入,对tRF的发生发展、作用机制以及在疾病中的潜在作用将会进一步丰富。拟从tRF作为新的基因表达调控分子的角度,简要介绍tRF发挥作用的分子机制。     

人线粒体tRNA修饰碱基与遗传性脑肌病

人的多种遗传疾病与线粒体tRNA基因突变有关,这些突变导致疾病发生的分子机理是当前研究的热点.通过研究线粒体tRNA分子上的碱基修饰情况,人们发现了一类特殊的带有牛磺酸衍生物基团的修饰,这类修饰主要位于线粒体tRNALys和线粒体tRNALeu(UUR)反密码子第一位摆动(wobble)位点的碱基上.最近的研究表明,位于这两种线粒体tRNA基因上的多种突变与遗传性脑肌病相关,包括A8344G,A3243G,T3271C等等,它们可以导致tRNA上相应摆动位点的碱基修饰缺失.无论是在体外培养的带有相应突变的细胞内,还是在来源于脑肌病病人的组织中,科学家都发现了相同的线粒体tRNA碱基修饰缺陷.通过分子手术证实,此类碱基修饰对于维持这两种tRNA的反密码子与mRNA上相应密码子的相互识别至关重要,缺失了这种修饰的tRNA将无法识别一些对应的密码子.通过进一步的实验,人们还鉴定出负责催化此类碱基修饰的酶.这些研究不但揭示了线粒体遗传性脑肌病相关突变的致病机理,也将为研究基因治疗提供可能的新手段.     

草菇基因组中的tRNA分析

草菇(V olvariella volvacea),又名中国蘑菇,是一种生长于中国和东南亚的重要经济食用菌。本文基于本实验室的草菇基因组测序结果,分析了草菇基因组中的tRNA情况。草菇基因组的302个框架中,共发现了177个tRNA基因,其中有7个可能的假基因,有2个携带硒代半胱氨酸,一个抑制性tRNA基因和一个未知的异型结构tRNA。除了上述11个特殊的tRNA基因外,166个tRNA可按反密码子类型分成47类。与其它5种担子菌的基因组tRNA比较,草菇的tRNA数量处于第4位,少于鬼伞、裂褶菌、灵芝,多于双孢蘑菇和平菇。通过比较分析草菇及这5种大型真菌的基因组tRNA编码情况,首次报道了几种食用菌tRNA遵守修正的摆动假说的情况。另外,草菇的同功受体tRNA非编码子序列也具有差异性,对tRNA的分析将有助于进一步研究草菇的进化。     

氨酰-tRNA合成酶对tRNA的识别

氨酰-tRNA合成酶(aaRS)与tRNA的相互作用保证了蛋白质生物合成的忠实性. 氨酰-tRNA合成酶对tRNA识别的专一性依赖于aaRS特定的催化结构域和tRNA分子特异的三级结构构象. 反密码子和接受茎(包括73位)在大多数aaRS对tRNA分子的识别过程中起着关键作用, 其他部位如可变口袋、可变(茎)环等, 甚至修饰核苷酸对于一些识别过程也有重要作用.     

The tRNA cycle and its relation to the rate of protein synthesis

With the aid of a kinetic model, we have investigated how the adaptation between the various components of the tRNA cycle and the codon frequencies affects the rate of protein synthesis. Depending on the relative amounts of total tRNA, synthetase and ribosomes, the optimal correlations vary between a situation where all tRNA species are either present in equal amounts or are present in amounts proportional to the square-root of the corresponding codon frequencies, and a situation where the amounts of the different tRNA species present are linearly proportional to the codon frequencies.Abbreviations EFTu Elongation factor Tu     

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate‐based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase‐coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell‐free protein synthesis activity. The resulting tRNA‐depleted lysate is compatible with in vitro‐transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real‐time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401–1407, 2017     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.     

Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria

Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the ‘antisense’ tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs.     

基于翻译调控的细菌耐药研究进展

由于抗生素的大量使用,细菌耐药问题凸显,直接威胁人类生命健康和世界经济发展。过去对于细菌耐药的遗传和分子机制研究较为透彻,而对应的调控机制研究相对较少。翻译调控作为生命体最重要的调控方式之一,在细菌耐药研究领域的重要性尚未被学术界充分重视。本文介绍了影响翻译过程的抗生素的主要作用机制,重点从核糖体的修饰和突变、tRNA总量的动态调控、tRNA氨酰化、tRNA甲基化、核糖体保护蛋白和翻译因子这几个方面概述了基于翻译调控的细菌耐药研究进展,为研究者们提供了一个基于翻译调控角度研究细菌耐药的新视角,同时也为开发靶向细菌翻译调控的新型抗生素提供一些新思路。     

Genes adopt non‐optimal codon usage to generate cell cycle‐dependent oscillations in protein levels

The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle‐regulated genes with that of other genes, we discovered that there is a significant preference for non‐optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non‐optimal codon preferences. Remarkably, cell cycle‐regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non‐optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl‐, threonyl‐, and glutamyl‐prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non‐optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell‐cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle‐dependent dynamics of proteins.     

Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs

The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.     

A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA.