共查询到19条相似文献,搜索用时 99 毫秒
1.
用T7 RNA聚合酶体外转录合成大鼠肝tRNAIle 总被引:1,自引:0,他引:1
采用PCR技术从rec-M13mp18中扩增出120 bp的大鼠肝tRNAIle合成基因片段,经限制性内切酶BstNⅠ酶切后作为模板,利用T7 RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNAIle基因,生成不含修饰碱基的tRNAIle,并对体外转录反应条件进行了优化,回收的tRNA产量可达DNA模板量的40倍. 相似文献
2.
为了研究tRNATrp的氨基酸接受茎中除两对半碱基以外的特异性元件,设计并完成了4种水稻线粒体tRNATrp向枯草杆菌tRNATrp的突变体 (MPB0, G1A和U5G/A68C;MPB1,C2G/G71C;MPB2,C4G/G69C;MPB3,C2G/G71C和C4G/G69C),体外转录并用枯草杆菌和人这两种不同种属来源的色氨酰 tRNA 合成酶(TrpRS)测定了这些 tRNATrp 分子的氨酰化活力(Kcat/KM).结果表明,这些突变体具有被枯草杆菌TrpRS氨酰化的能力,与野生型水稻线粒体tRNATrp相比,MPB0被枯草杆菌TrpRS氨酰化的活力提高了5倍,MPB1和MPB2被枯草杆菌TrpRS氨酰化的活力分别提高了40和53倍,MPB3则提高了140倍,为野生型枯草杆菌tRNATrp的34%,而人色氨酰 tRNA合成酶氨酰化这4个突变体的活力都很微弱.揭示了水稻线粒体tRNATrp氨基酸接受茎上的2个碱基对C2/G71和C4/G69的突变,对枯草杆菌TrpRS的识别起重要作用,由此推测,接受茎上的2个碱基对C2/G71和C4/G69也是线粒体tRNATrp重要的特异性元件. 相似文献
3.
大肠杆菌tRNALeu的基因克隆、高效表达和纯化 总被引:2,自引:0,他引:2
用化学法合成的tRNALeu 和tRNALeu 2的基因分别连接到 pTrc99B质粒载体上 ,转化到大肠杆菌MT10 2中 .DNA测序筛选得到与已知tRNALeu1 和tRNALeu2 的基因顺序完全相同的克隆 .对带有tRNALeu1 和tRNALeu2 基因的 2个转化子 (MT -Leu1和MT- Leu2 )表达条件进行了优化 ,MT- Leu1和MT- Leu2总tRNA中的亮氨酸接受活力分别达到 810 pmol/A2 6 0 和 5 60 pmol/A2 6 0 :tRNALeu1 占MT -Leu1总tRNA的5 0 % ;tRNALeu2 占MT- Leu2总tRNA的 3 0 % .经DEAE Sepharose、BD -纤维素层析柱 ,可分别将MT- Leu1和MT -Leu2的总tRNA纯化到 160 0pmol/A2 6 0 .首次准确地测得了 2种等受体tRNALeu的氨酰化反应动力学常数 . 相似文献
4.
用化学方法合成编码 2个大肠杆菌tRNALeu(tRNALeu1和tRNALeu2 )的基因和T7启动子 ,分别克隆到pUC1 9载体上 ,并在纯化的T7RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNALeu.在T7转录体系中 ,亚精胺对转录有负影响 .在最适转录条件下 ,可以得到有活力的RNA转录物的量是模板DNA的 2 5 0倍左右 .在大肠杆菌亮氨酰 tRNA合成酶的催化下 ,2种经体外转录产生的未修饰等受体tRNALeu(tRNALeu1和tRNALeu2 )的亮氨酸接受能力基本相同 ,但只有从体内纯化对应的tRNALeu的四分之一左右 ,表明修饰核苷酸在tRNALeu氨酰化过程中起着较为重要但非关键的作用 . 相似文献
5.
酵母tRNAAla的3′半分子与一个11聚的DNA片段(5′GGAATCGAACC3′)杂交后用RNase H酶解,该酶能在Ψ55的3′侧定点剪切,这样就制备得片段C36-Ψ55该片段经1~2个高碘酸氧化和β-消去得片段C36-T54和C36-G53机器合成了3个酵母tRNAAla的片段,分别j是片段C56-A76,U55-A76(以U替代Ψ55)和U54-A76(以UU替代T54Ψ55).合成和制备的片段以适当的组合用T4RNA连接酶连接,产物是酵母tRNAtRNAAla的3′半分子或其类似物.3种3′半分子或其类似物分别与天然5′半分子连接得重组天然酵母tRNAAla(tRNAr)和2个酵母tRNAAla的类似物:(1)tRNAa(以U替代Ψ55),(2)tRNAb(以UU替代T54Ψ55).体外测定了它们的丙氨酸接受活力和参入活力,发现酵母tRNAAla的类似物tRNAa和tRNAb与天然重组酵母tRNAAla相比,它们的氨基酸接受活力分别降低了25%和55%,参入活力分别降低了35%和30%.说明酵母tRNAAla中的修饰核苷酸T54和Ψ55对该tRNA的功能有重要的影响. 相似文献
6.
p16INK4a基因的功能及其调控 总被引:1,自引:0,他引:1
p16INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16INK4a对某些肿瘤进行基因治疗的研究正在进行中. 相似文献
7.
线粒体tRNA基因突变是导致感音神经性耳聋的原因之一.有些tRNA突变可直接造成耳聋的发生,称之为原发突变.如tRNALeu(UUR) A3243G等突变与综合征型耳聋相关,而tRNASer(UCN) T7511C等突变则与非综合征型耳聋相关.此外,继发突变如tRNAThr G15927A等突变则对原发突变起协同作用,影响耳聋的表型表达.这些突变可引起tRNA二级结构改变,从而影响线粒体蛋白质合成,降低细胞内ATP的产生,由此引起的线粒体功能障碍可导致耳聋的发生.主要讨论与耳聋相关的线粒体tRNA突变及其致聋机理. 相似文献
8.
本文报道一种E.coli tRNALeu简便而稳定的纯化方法。粗tRNA经过BD-Cellulose柱层析和聚丙烯酰胺凝胶电泳两个步骤即可得到亮氨酸接受能力为1400pmol/A260单位的tRNALeu。 相似文献
9.
p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探 总被引:2,自引:0,他引:2
ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14ARF高表达能将细胞周期阻断在G1和G2期, p53, p21cip1和p27kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖. 相似文献
10.
揭示了吖啶橙的吸收光谱和荧光光谱对其浓度依赖性上的区域性特征,分析了测定溶酶体H+转运时合理选用吖啶橙浓度及溶酶体用量的重要性、机理和原则,探讨了其与溶酶体的温育时间和K+/H+交换对测定H+转运的明显影响. 相似文献
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12.
Li Z Liu Y Yang L Wang S Guan MX 《Biochemical and biophysical research communications》2008,367(4):906-911
Mutations in mitochondrial DNA have been associated with cardiovascular disease. We report here the clinical, genetic, and molecular characterization of one three-generation Han Chinese family with maternally transmitted hypertension. All matrilineal relatives in this family exhibited the variable degree of hypertension at the age at onset of 36 to 56 years old. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the known hypertension-associated tRNAIle A4295G mutation and 33 other variants, belonging to the Asian haplogroup D4j. The A4295G mutation, which is extraordinarily conserved from bacteria to human mitochondria, is located at immediately 3′ end to the anticodon, corresponding to conventional position 37 of tRNAIle. The occurrence of the A4295G mutation in several genetically unrelated pedigrees affected by cardiovascular disease but the absence of 242 Chinese controls strongly indicates that this mutation is involved in the pathogenesis of cardiovascular disease. Of other variants, the tRNAGlu A14693G and ND1 G11696A mutations were implicated to be associated with other mitochondrial disorders. The A14693G mutation, which is a highly conserved nucleoside at the TψC-loop of tRNAGlu, has been implicated to be important for tRNA structure and function. Furthermore, the ND4 G11696A mutation was associated with Leber’s hereditary optic neuropathy. Therefore, the combination of the A4295G mutation in the tRNAIle gene with the ND4 G11696A mutation and tRNAGlu A14693G mutation may contribute to the high penetrance of hypertension in this Chinese family. 相似文献
13.
Pathology-related substitutions in human mitochondrial tRNA(Ile) reduce precursor 3' end processing efficiency in vitro
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The human mitochondrial genome encodes 22 tRNAs interspersed among the two rRNAs and 11 mRNAs, often without spacers, suggesting that tRNAs must be efficiently excised. Numerous maternally transmitted diseases and syndromes arise from mutations in mitochondrial tRNAs, likely due to defect(s) in tRNA metabolism. We have systematically explored the effect of pathogenic mutations on tRNAIle precursor 3′ end maturation in vitro by 3′-tRNase. Strikingly, four pathogenic tRNAIle mutations reduce 3′-tRNase processing efficiency (Vmax / KM) to ~10-fold below that of wild-type, principally due to lower Vmax. The structural impact of mutations was sought by secondary structure probing and wild-type tRNAIle precursor was found to fold into a canonical cloverleaf. Among the mutant tRNAIle precursors with the greatest 3′ end processing deficiencies, only G4309A displays a secondary structure substantially different from wild-type, with changes in the T domain proximal to the substitution. Reduced efficiency of tRNAIle precursor 3′ end processing, in one case associated with structural perturbations, could thus contribute to human mitochondrial diseases caused by mutant tRNAs. 相似文献
14.
Caroline K?hrer Debabrata Mandal Kirk W. Gaston Henri Grosjean Patrick A. Limbach Uttam L. RajBhandary 《Nucleic acids research》2014,42(3):1904-1915
Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2Ile to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNAIle-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2Ile is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1Ile, in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain. 相似文献
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16.
《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1988,950(2):172-181
Transfer RNA from Escherichia coli C6, a Met−, Cys−, relA− mutant, was previously shown to contain an altered tRNAIle which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNAIle and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNAIle purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNAIle (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNAIle bound more efficiently to its synthetase compared to normal tRNAIle. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNAIle contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNAIle contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNAIle without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNAIle has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNAIle from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNAIle for aminoacylation is discussed. 相似文献
17.
In the bacterial decoding system, the AUA codon is deciphered as isoleucine by tRNAIle bearing lysidine (L, 2-lysyl-cytidine) at the wobble position. Lysidine is an essential modification that determines both the codon and amino acid specificities of tRNAIle. We identified an enzyme named tRNAIle lysidine synthetase (TilS) that catalyzes lysidine formation by using lysine and ATP as substrates. Biochemical studies revealed a molecular mechanism of lysidine formation that consists of two consecutive reactions involving the adenylated tRNA intermediate. In addition, we deciphered how Escherichia coli TilS specifically discriminates between tRNAIle and the structurally similar tRNAMet, which bears the same anticodon loop. Recent structural studies unveiled tRNA recognition by TilS, and a molecular basis of lysidine formation at atomic resolution. 相似文献
18.
T. Niimi G. Kawai M. Takayanagi T. Noguchi N. Hayashi T. Kohno Y. Muto K. Watanabe T. Miyazawa S. Yokoyama 《Biochimie》1993,75(12)
Imino 15N and 1H resonances of Escherichia coli tRNAlIle were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNAlIle with isoleucyl-tRNA synthetase, some imino 15N-1H resonances disappeared, and some others were significantly broadened and/or shifted in the 1H chemical shift, while the others were observed at the same 15H-1H chemical shifts. It was indicated that the binding of tRNAlIle with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNAlIle by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNAlIle molecule, which induces changes in the secondary and tertiary structures of tRNAlIle. 相似文献
19.
Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNAIle is present in ≤ 30% the amount of tRNAThr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNAThr and tRNAIle, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNAThr + Ile is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNAIle can be explained by a novel processing pathway that generates a metabolically unstable tRNAIle product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNAIle has a unique physiological role. 相似文献