ACTIONS OF THE ANTICODON ARM IN TRANSLATION ON THE PHENOTYPES OF RNA MUTANTS
ACTIONS OF THE ANTICODON ARM IN TRANSLATION ON THE PHENOTYPES OF RNA MUTANTS
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DOI：
10.1016/0022-2836(86)90362-1
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发表时间：
1986-11-20
影响因子：
5.6
通讯作者：
THOMPSON, RC
中科院分区：
文献类型：
--
作者：
YARUS, M;CLINE, SW;THOMPSON, RC
在以前的出版物中，我们已经表明，它是实用的研究的翻译活性的tRNAs的质粒克隆上的属的反密码子臂序列的替换和改变。将反密码子臂序列移植到tRNA基因之间的实验表明，翻译活性由这些序列决定。因此，我们制作了反密码子环的每一个变体和靠近环的茎的三个碱基对，以解决Su 7 Am tRNA Trp的结构与其功能之间的关系。所有衍生物都保留了分子的正常二级结构，这是已知的翻译活性所必需的。在这项工作中测量的琥珀密码子的翻译，这些抑制剂的概率。这种体内翻译活性根据质粒克隆的拷贝数、tRNA的核苷酸修饰、成熟tRNA的稳态水平和这些分子的氨酰化的数据来合理化。这些tRNA的核苷酸修饰水平各不相同，提供了关于修饰系统特异性的信息，这些修饰系统使O-甲基核糖、假尿苷和反密码子臂中的修饰A。然而，对于这一系列tRNA，这些修饰对tRNA的翻译效率没有强烈的影响。通过比较正常菌株和相关菌株中的抑制，确定了一些取代减少了谷氨酰胺对tRNA的氨酰化，这些菌株具有25倍升高水平的谷氨酰胺酰-tRNA合成酶（GlnRS）。出乎意料的是，对GlnRS作用影响最大的取代是反密码子环5′侧上保守嘧啶的嘌呤。体内tRNA浓度的数据表明，反密码子环和螺旋有助于类似的确定稳态水平的tRNA。尽管所有的tRNA都是由相同的转录单位制成的同源前体加工而成，但这种水平变化了七倍。对水平的影响似乎是由反密码子臂结构的变化介导的。为了解决对tRNA水平和核糖体步骤的影响，开发了一个将氨酰-tRNA水平与抑制剂效率相关联的稳健方程：E= A（K+ A），其中E是效率，A是氨酰-tRNA浓度，K是有效浓度，或单个tRNA具有0.50的效率所需的细胞tRNA含量。在其他影响被标准化之后，tRNA在其对核糖体的内在功能（由K表示）方面有所不同。环序列的翻译活性变化超过1000倍，较小的两倍效应可能归因于螺旋序列和结构的变化。环位置37和38是核糖体步骤效率的最重要决定因素。这种环模式与核糖体上3′堆叠环的作用一致，就像晶体结构中的堆叠一样。天然反密码子环区的序列对于翻译效率是最佳的。所有变体的活性都低于亲本Su 7 tRNA中存在的活性，其最类似于翻译密码子UNN的天然延伸体tRNA。天然环序列优化了tRNA分子的翻译功能，尽管它使tRNA水平最小化。在其他密码子的tRNA中经常发现的序列，当引入Su 7时，大大降低了这种tRNA的效率。这种行为与扩展反密码子假说一致。与环核苷酸相反，螺旋核苷酸对主要通过tRNA水平影响翻译效率。天然螺旋序列...
In previous publications, we have shown that it is practical to study the translational activity of tRNAs by replacement and alteration of the anticodon arm sequence of the genus on a plasmid clone. Experiments in which the anticodon arm sequence is transplanted between tRNA genes suggest that the translational activity is determined by these sequences. We have therefore made every variant of the anticodon loop and the three base-pairs of the stem proximal to the loop, in order to resolve the relation between the structure of Su7 Am tRNA Trp, and its function. All derivatives conserved the normal secondary structure of the molecule, which was known to be essential for translational activity. The probability of translation of the amber codon by these suppressors is measured in this work. This translational activity in vivo is rationalized in terms of data on the copy numbers of the plasmid clones, the nucleotide modifications of the tRNAs, the steady-state level of the mature tRNA, and the aminoacylation of these molecules. Nucleotide modification levels vary among these tRNAs, giving information about the specificities of modification systems that make O-methylribose, pseudouridine, and modified A in the anticodon arm. However, for this series of tRNAs, none of these modifications has a strong effect on translational efficiency of the tRNAs. A few of the substitutions reduce aminoacylation of the tRNAs with glutamine, as determined by comparison of suppression in normal strains and related strains, which have 25-fold elevated levels of the glutaminyl-tRNA synthetase (GlnRS). The substitutions that have the largest effect on GlnRS action are, unexpectedly, purines for conserved pyrimidines on the 5′ side of the anticodon loop. Data on the concentrations of tRNA in vivo suggest that the anticodon loop and helix contribute similarly to the determination of the steady-state level of the tRNAs. This level varies sevenfold, though all tRNAs are processed from a homologous precursor made from the same transcription unit. Effects on levels appear to be mediated by changes in anticodon arm structure. A robust equation that relates aminoacyl-tRNA levels to suppressor efficiency is developed in order to resolve effects on tRNA levels and on ribosomal steps: E= A (K+ A), where E is efficiency, A is aminoacyl-tRNA concentration, and K is the effective concentration, or cellular tRNA content required for an individual tRNA to have an efficiency of 0.50. The tRNAs vary in their intrinsic ability to function on the ribosome (represented by K), after other influences have been normalized. Loop sequences vary more than 1000-fold in translational activity, and smaller, twofold effects may be attributed to variation of the helix sequence and structure. Loop positions 37 and 38 are the most important determinations of the efficiency of the ribosomal steps. This loop pattern is consistent with the action of a 3′ stacked loop on the ribosome, like the stack in the crystallographic structure. The sequence of the natural anticodon loop region is optimal for translational efficiency. All variants are less active than that present in the parental Su7 tRNA, which most resembles a natural elongator tRNA that translates codon UNN. The natural loop sequence optimizes the translational function of the tRNA molecule in spite of the fact that it minimizes the tRNA level. Sequences frequently found in tRNAs for other codons, when introduced into Su7, substantially reduce the efficiency of this tRNA. This behavior is consistent with the extended anticodon hypothesis. In contrast to loop nucleotides, helix nucleotide pairs influence translational efficiency mainly through tRNA levels. The natural helix sequence …