An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.     

Aminoacylation of synthetic DNAs corresponding to Escherichia coli phenylalanine and lysine tRNAs

Synthetic DNA oligomers (tDNAs) corresponding to Escherichia coli tRNA(Phe) or tRNA(Lys) have been synthesized with either deoxythymidine (dT) or deoxyuridine (dU) substituted in the positions occupied by ribouridine or its derivatives. The tDNAs inhibited the aminoacylation of their respective tRNAs with their cognate amino acids, but not the aminoacylation of tRNA(Leu) with Leu. In the presence of aminoacyl-tRNA synthetase, species of both a tDNA(Phe) synthesized with a 3' terminal riboadenosine and a tDNA(Lys) containing only deoxynucleotides could be aminoacylated with the appropriate amino acids, although the Michaelis constant Km and observed maximal rate Vmax values for aminoacylation were increased by three- to fourfold and decreased by two- to threefold, respectively. The aminoacylation of synthetic tDNAs demonstrates that the ribose backbone of a tRNA is not absolutely required for tRNA aminoacylation.     

Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine

The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.     

Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA

Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNA(CUA) with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.     

Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation

Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis. Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body. The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated. Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.     

Virus attenuation by genome-scale changes in codon pair bias

As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.     

Polypeptide sequences essential for RNA recognition by an enzyme

Many RNAs are complex, globular molecules formed from elements of secondary and tertiary structure analogous to those found in proteins. Little is known about recognition of RNAs by proteins. In the case of transfer RNAs (tRNAs), considerable evidence suggests that elements dispersed in both the one- and three-dimensional structure are important for recognition by aminoacyl tRNA synthetases. Fragments of alanine tRNA synthetase were created by in vitro manipulations of the cloned alaS gene and examined for their interaction with alanine-specific tRNA. Sequences essential for recognition were located near the middle of the polypeptide, juxtaposed to the carboxyl-terminal side of the domain for aminoacyl adenylate synthesis. The most essential part of the tRNA interaction strength and specificity was dependent on a sequence of fewer than 100 amino acids. Within this sequence, and in the context of the proper conformation, a segment of no more than 17 amino acids was responsible for 25% or more of the total synthetase-tRNA free energy of association. The results raise the possibility that an important part of specific RNA recognition by an aminoacyl tRNA synthetase involves a polypeptide segment that is short relative to the total size of the protein.     

Enzymatic modification of transfer RNA

The molecular events leading to the synthesis of mature tRNA are only now becoming amenable to experimental study. In bacterial and mammalian cells tRNA genes are transcribed into precursor tRNA. These molecules, when isolated, contain additional nucleotides at both ends (20) of the mature tRNA and lack most modified nucleosides. Presumably, specific nucleases ("trimming" enzymes) cut the precursor to proper tRNA size. The C-C-A nucleotide sequence of the amino acid acceptor end common to all tRNA's does not seem to be coded by tRNA genes (30), and may be added to the trimmed molecules by the tRNA-CMP-AMP-pyrophosphorylase (71). Modifications at the polynucleotide level of the heterocyclic bases or the sugar residues give rise to the modified nucleosides in tRNA. Although newly available substrates have allowed the detection of more of the enzymes involved in these reactions, there is still no knowledge about the sequence of modification or trimming events leading to the synthesis of active tRNA. Progress in these studies may not be easy because enzyme preparations free of nucleases or other tRNA modifying enzymes are required. The role of the modified nucleosides in the biological functions of tRNA is still unknown. Possibly pseudouridine is required for ribosome mediated protein synthesis; some other modified nucleosides in tRNA are not required for this reaction, but may enhance its rate. What might be the role of the large variety of modified nucleosides in tRNA? One is tempted to speculate that such nucleosides are important in other cellular processes in which tRNA is thought to participate such as virus infection, cell differentiation, and hormone action (2, 3). Mutants in a number of tRNA-modifying enzymes are needed in order to extend our knowledge of their purpose and of tRNA involvement in other biological processes. But unless tRNA-modifying enzymes specific for a particular tRNA species exist, no simple selection procedure can be devised. Possibly some of the regulatory mutants of amino acid biosynthesis may prove to affect tRNA-modifying enzymes (72). Transfer RNA's are macromolecules well suited for the study of nucleic acid-protein interactions. The tRNA molecules are structurally very similar, and they interact with a large number of enzymes or protein factors (2, 3). Each aminoacyl-tRNA synthetase, for instance, very precisely recognizes a set of cognate isoacceptor tRNA's (2, 73). The availability of the tRNA- modifying enzymes adds another dimension to the problem of the nature of specific recognition of tRNA by proteins. There are some tRNA-modifying enzymes, such as the uracil-tRNA methylase, which may recognize all tRNA species, while others, such as the isopentenyl-tRNA transferase, probably recognize only a selected set of tRNA molecules, even with different amino acid accepting capacities. With well-characterized RNA precursor and tRNA molecules we can hope to delineate those features of primary, secondary, and tertiary structure involved in the specific interactions of tRNA with these enzymes.     

小麦抗感白粉病品种游离氨基酸含量的比较分析

测定7个小麦抗感白粉病品种苗期、11个品种成株期叶片的游离氨基酸含量。结果表明,苗期抗病品种叶片各游离氨基酸中,缬氨酸、苯丙氨酸、组氨酸和精氨酸平均含量明显高于感病品种,与病情指数呈明显负相关,而其它12种游离氨基酸平均含量抗感品种间无明显差异。成株期倒2叶游离氨基酸中,缬氨酸、甲硫氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、赖氨酸、组氨酸、色氨酸、精氨酸及总游离氨基酸含量,均显著低于感病品种,且与病情指数呈明显正相关。各游离氨基酸平均含量苗期和成株期抗感品种间差异均不一致。     

Synonymous codon usage pattern in model legume Medicago truncatula

Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences (CDSs) from M. truncatula were examined for codon usage bias (CUB). Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.     

茶树脂肪氧合酶(LOX)基因家族成员的分子进化及密码子偏好性分析

脂肪氧合酶(lipoxygenase,LOX,EC 1.13.11.12)是一类含有铁离子的血红素双加氧酶,专一催化多元不饱和脂肪酸加氧反应。基于茶树基因组数据,对筛选获得的12个LOX基因家族成员的分子特性、进化规律及密码子偏好性进行分析归纳。理化性质检测结果表明,茶树LOX家族成员中以Cs LOX6的开放阅读框(open reading frame,ORFs)最长(2 778 bp),编码925个氨基酸,蛋白分子量为104.5 k Da。通过氨基酸序列同源比对,构建茶树LOX基因家族成员与26个物种(均系双子叶植物纲)的37个LOX基因的系统发育进化树,结果发现,茶树LOX基因家族成员可被分为9-LOX亚族(5个)、13-LOX TypeⅠ亚类(5个)及13-LOX TypeⅡ亚类(2个),但13-LOX TypeⅡ亚类中的Cs LOX5基因,并不具有转运肽(transit peptide)前导序列。对茶树基因组LOX家族成员基因密码子相对偏好使用度(relative synonymous codon usage,RSCU)及偏好性参数进行分析,结果表明:茶树LOX基因家族成员存在着4个密码子在编码基因上被高频率选用,9个密码子被低频率选用;茶树LOX基因家族成员的密码子选用偏好性普遍较弱,并偏好以A/T作为结尾。此外,以最近邻元素法(Neighbor-Joining,NJ)对密码子RSCU值进行聚类分析,结果发现,RSCU值的聚类与以编码序列(coding sequences,CDS)构建的系统发育进化树之间,在Cs LOX10基因的聚类上存在差异。     

Model substrates for an RNA enzyme

M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence. The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop. The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.     

Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end

Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.     

TYK2基因与家兔肠炎的易感性研究

[目的]研究TYK2基因在家兔肠炎发生中的遗传效应,探讨其是否可以作为家兔抗病育种的辅助选择标记。[方法]通过构建病例组-对照组肠炎兔群和低纤维诱导的肠炎兔群,PCR产物纯化后直接测序,以及qRT-PCR法检测TYK2基因在回肠和结肠组织中的mRNA表达水平。[结果]发现TYK2基因外显子区域有5个c SNP,只有c.1477(c.1477,CT)位点发生非同义突变,导致氨基酸p.Leu 404 Phe(LF)的改变,C等位基因增加了肠炎的易感性(OR:1.36;95%CI 1.269~2.151;P=0.019),而等位基因T对肠炎的发生起到了一定的保护作用(OR:0.81,95%CI 0.352~0.960;P=0.018)。TYK2基因不同基因型和不同肠炎程度的mRNA水平均呈现差异表达(P0.05)。[结论]TYK2(c.1477,CT)是家兔肠炎易感风险基因,为家兔的抗病育种提供了一个可靠的辅助选择标记。     

影响伪狂犬病病毒同义密码子用法特点的因素分析

为了充分了解伪狂犬病病毒基因组结构和病毒进化机制,计算伪狂犬病病毒各基因(组)Nc值、RSCU值、GC3s含量和双核苷酸组成,分析伪狂犬痛病毒密码子用法特点.采用目前最普遍使用的多变量统计分析方法(对应分析)分析影响伪狂犬病病毒基因组同义密码子用法偏爱性的因素.结果表明:①在GC含量丰富的伪狂犬病病毒基因组中,所有基因都偏爱于以G或C结尾的密码子;②伪狂犬病病毒对CpG、CpC、GpC和GpG 4种双核苷酸具有显著偏爱性,而较少使用ApA、ApT、TpA和TpT 4种双核苷酸;③碱基组成限制、碱基突变压力、翻译选择和基因功能是形成伪狂犬病病毒密码子用法特点的4种因素.伪狂犬病病毒所有基因Nc-GC3s分布图显示有些基因如LLT ORF1、LLT ORF2等的偏向性完全是由于碱基的组成限制.GC3s-GC12s散点图则显示碱基突变和自然选择都是PRV密码子偏向性的形成因素.根据RSCU值进行的对应分析表明伪狂犬病病毒大多数基因密码子用法受基因表达水平和基因功能影响.综上所述,伪狂犬病病毒偏爱于G或C结尾的密码子,且碱基组成限制、碱基突变、翻译选择和基因功能是影响伪狂犬病病毒同义密码子用法特点的主要因素.     

甘南牦牛H-FABP基因CDS区多态性及生物信息学分析

应用PCR产物混合样本DNA池法检测甘南牦牛心脏型脂肪酸结合蛋白(H-FABP)基因CDS区多态性,并应用生物信息学方法分析甘南牦牛H-FABP蛋白质特性.结果表明:甘南牦牛H-FABP基因CDS区序列与九龙牦牛相同,而与普通牛对比在第3外显子存在*76G>A的同义突变;甘南牦牛H-FABP氨基酸序列没有明显的疏水性区域,也未形成跨膜螺旋区及信号肽,推测其主要在细胞质中发挥生物学作用;甘南牦牛H-FABP基因编码产物二级结构是以α-螺旋和β-折叠为主的mixed型;氨基酸序列与普通牛、山羊、马、人、小鼠、大鼠、鸡、草雀及绿鸭9个物种间同源性较高,与其实际亲缘关系远近一致.     

玉米叶绿体基因密码子使用频率分析

本研究应用软件CodonW,对玉米叶绿体蛋白编码基因的密码子用法进行了初步分析,计算了位于密码子3个位置的GC含量和同义密码子的使用频率,并确定了玉米叶绿体基因组的最优密码子。结果显示玉米叶绿体基因GC含量低,密码子第三位的GC含量最低,偏好使用以A或T碱基结尾的密码子;且确定的25种最优密码子也主要以A或T碱基结尾。分析结果对指导外源基因进行分子改造,提高其在玉米叶绿体中的表达效率及细胞器分子进化研究等具有重要意义。     

玉米同义密码子使用偏性分析

利用玉米B73全基因组53 764个基因的表达序列数据,使用多重变量分析软件CodonW对影响密码子用法的因素进行分析。结果表明:在59种同义密码子中有27种为玉米最优密码子。同时还指出,最优密码子的使用频率(FOP)与基因的G+C含量(GC),尤其是第3位密码子的G+C百分含量(GC3S)、密码子适应指数(CAI)和密码子偏爱指数(CBI)之间均呈现极显著的正相关(相关系数分别为r=0.780 7、0.822 3、0.731 4和0.986 3),而与有效等位基因数ENc存在显著负相关(相关系数r=-0.681 5)。表明基因GC含量直接影响玉米密码子使用的偏性,同时基因的表达水平越高,对密码子的使用偏向性越强。27个最优密码子的首次确定将对玉米转基因具有重要指导意义。     

马麝朊蛋白(PrP)基因的克隆和序列分析

从马麝的全血中提取基因组总DNA,用所设计引物以聚合酶链式反应扩增出细胞型朊蛋白(PrPC)基因,并克隆到pMD18-T载体。序列分析表明所克隆的马麝PrP基因片段大约为771bp,包含了朊蛋白基因的完整编码区序列,即包含在单一外显子内的完整开放阅读框,与国外报道的同科动物PrP基因序列基本相同。马麝PrP基因含5个短而富含G-C的元件,可编码5个八肽(九肽)重复Pro-His-Gly-Gly-Gly-Trp-Gly-Gln或Pro-Gln/His-Gly-Ala/Gly-Gly–Gly-Trp-Gly-Gln,其氨基酸序列含有24个氨基酸的N-端信号肽和23个氨基酸的C-端信号肽。与白尾鹿(Odocoileusvirginianus)和麋鹿(Cervuselaphus)的PrP基因相比,其核苷酸序列和推导氨基酸序列同源性分别为97.4%、97.9%和98.1%、97.7%。共发生15个碱基替换,其中10个为同义码替换,5个为异义突变,即G57A、S100N、N173S、T177N和M208I。